The effect of normobaric hypoxia on acute exercise-induced changes in blood sphingoid base-1-phosphates metabolism in cyclists.

Extracellular sphingosine-1-phosphate (S1P) emerged as an important regulator of muscle function. We previously found that plasma S1P concentration is elevated in response to acute exercise and training. Interestingly, hypoxia, which is commonly utilized in training programs, induces a similar effect. Therefore, the aim of the current study was to determine the effect of normobaric hypoxia on exercise-induced changes in blood sphingolipid metabolism. Fifteen male competitive cyclists performed a graded cycling exercise until exhaustion (GE) and a simulated 30 km individual time trial (TT) in either normoxic or hypoxic (FiO2 = 16.5%) conditions. Blood samples were taken before the exercise, following its cessation, and after 30 min of recovery. We found that TT increased dihydrosphingosine-1-phosphate (dhS1P) concentration in plasma (both HDL- and albumin-bound) and blood cells, as well as the rate of dhS1P release from erythrocytes, regardless of oxygen availability. Plasma concentration of S1P was, however, reduced during the recovery phase, and this trend was augmented by hypoxia. On the other hand, GE in normoxia induced a selective increase in HDL-bound S1P. This effect disappeared when the exercise was performed in hypoxia, and it was associated with reduced S1P level in platelets and erythrocytes. We conclude that submaximal exercise elevates total plasma dhS1P concentration via increased availability of dihydrosphingosine resulting in enhanced dhS1P synthesis and release by blood cells. Maximal exercise, on the other hand, induces a selective increase in HDL-bound S1P, which is a consequence of mechanisms not related to blood cells. We also conclude that hypoxia reduces post-exercise plasma S1P concentration.

Hepcidin and erythroferrone response to 3 weeks of exposure to normobaric hypoxia at rest in trained cyclists.

Purpose: The effectiveness of altitude training on haematological adaptations is largely dependent on iron metabolism. Hepcidin and erythroferrone (ERFE) are key iron-regulating hormones, yet their response to altitude training is poorly understood. The aim of this study was to analyze changes in hepcidin and ERFE under the influence of 3 weeks of the Live High-Train Low (LH-TL) method. Methods: Twenty male trained cyclists completed a 3-week training program under normoxic conditions (NORM) or with passive exposure to normobaric hypoxia (LH-TL; FiO2 = 16.5%, ∼2000 m; 11-12 h/day). Hepcidin, ERFE, hypoxia inducible factor-2 (HIF-2), ferroportin (Fpn), erythropoietin (EPO), serum iron (Fe) and hematological variables were assessed at baseline (S1), then immediately after (S2) and 3 days after (S3) intervention. Results: In the LH-TL group, hepcidin decreased by 13.0% (p < 0.001) in S2 and remained at a reduced level in S3. ERFE decreased by 28.7% (p < 0.05) in S2 and returned to baseline in S3. HIF-2α decreased gradually, being lower by 25.3% (p < 0.05) in S3. Fpn decreased between S1 and S2 by 18.9% (p < 0.01) and remained lower during S3 (p < 0.01). In the NORM group, in turn, hepcidin levels increased gradually, being higher by 73.9% (p < 0.05) in S3 compared to S1. No statistically significant differences in EPO were observed in both groups. Conclusion: Three weeks of LH-TL suppresses resting hepcidin and ERFE levels in endurance athletes. We found no association between hepcidin and ERFE after LH-TL. Probably, ERFE is not the only factor that suppresses hepcidin expression in response to moderate hypoxia, especially in later stages of hepcidin downregulation. With the cessation of hypoxia, favorable conditions for increasing the availability of iron cease.

Evaluation of Oromucosal Natural Gum-Based Emulgels as Novel Strategy for Photodynamic Therapy of Oral Premalignant Lesions.

Photodynamic therapy (PDT) recently has been shown as a promising option in the treatment of premalignant lesions of the soft oral tissues. Effective delivery of photosensitizer is challenging due to poor drug adherence to the oromucosal epithelium. In the present work, emulgels composed of natural polysaccharide gums (tragacanth, xanthan and gellan) were evaluated as novel oromucosal platforms of delta-aminolevulinic acid (ALA) for PDT. Apart from mucoadhesive and textural analysis, the specific steps involved studies on drug penetration behavior and safety profile using a three-dimensional human oral epithelium model (HOE). All designed emulgels presented greater mucoadhesiveness when compared to commercial oromucosal gel. Incorporation of ALA affected textural properties of emulgels, and tragacanth/xanthan formulation with greater hardness and cohesiveness exhibited a protective function against the mechanical tongue stress. Permeability studies revealed that ALA is capable of penetrating across oromucosal epithelium by passive transport and all formulations promoted its absorption rate when compared to a commercial topical product with ALA. Importantly, the combination of tragacanth and xanthan profoundly enhanced photosensitizer retention in the buccal epithelium. Tested samples performed negligible reduction in cell viability and moderately low IL-1ß release, confirming their non-irritancy and compatibility with HOE. Overall, the presented findings indicate that tragacanth/xanthan emulgel holds promise as an oromucosal ALA-carrier for PDT strategy.

Effect of Normobaric Hypoxia on Alterations in Redox Homeostasis, Nitrosative Stress, Inflammation, and Lysosomal Function following Acute Physical Exercise.

Hypoxia is a recognized inducer of oxidative stress during prolonged physical activity. Nevertheless, previous studies have not systematically examined the effects of normoxia and hypoxia during acute physical exercise. The study is aimed at evaluating the relationship between enzymatic and nonenzymatic antioxidant barrier, total antioxidant/oxidant status, oxidative and nitrosative damage, inflammation, and lysosomal function in different acute exercise protocols under normoxia and hypoxia. Fifteen competitive athletes were recruited for the study. They were subjected to two types of acute cycling exercise with different intensities and durations: graded exercise until exhaustion (GE) and simulated 30 km individual time trial (TT). Both exercise protocols were performed under normoxic and hypoxic (FiO2 = 16.5%) conditions. The number of subjects was determined based on our previous experiment, assuming the test power = 0.8 and α = 0.05. We demonstrated enhanced enzymatic antioxidant systems during hypoxic exercise (GE: ↑ catalase (CAT), ↑ superoxide dismutase; TT: ↑ CAT) with a concomitant decrease in plasma reduced glutathione. In athletes exercising in hypoxia, redox status was shifted in favor of oxidation reactions (GE: ↑ total oxidant status, ↓ redox ratio), leading to increased oxidation/nitration of proteins (GE: ↑ advanced oxidation protein products (AOPP), ↑ ischemia-modified albumin, ↑ 3-nitrotyrosine, ↑ S-nitrosothiols; TT: ↑ AOPP) and lipids (GE: ↑ malondialdehyde). Concentrations of nitric oxide and its metabolites (peroxynitrite) were significantly higher in the plasma of hypoxic exercisers with an associated increase in inflammatory mediators (GE: ↑ myeloperoxidase, ↑ tumor necrosis factor-alpha) and lysosomal exoglycosidase activity (GE: ↑ N-acetyl-ß-hexosaminidase, ↑ ß-glucuronidase). Our study indicates that even a single intensive exercise session disrupts the antioxidant barrier and leads to increased oxidative and nitrosative damage at the systemic level. High-intensity exercise until exhaustion (GE) alters redox homeostasis more than the less intense exercise (TT, near the anaerobic threshold) of longer duration (20.2 ± 1.9 min vs. 61.1 ± 5.4 min-normoxia; 18.0 ± 1.9 min vs. 63.7 ± 3.0 min-hypoxia), while hypoxia significantly exacerbates oxidative stress, inflammation, and lysosomal dysfunction in athletic subjects.

Grade-dependent changes in sphingolipid metabolism in clear cell renal cell carcinoma.

There is a host of evidence for the role of bioactive sphingolipids in cancer biology, and dysregulated sphingolipid metabolism was observed in many malignant tumors. The aim of the present study was to provide more detailed data on sphingolipid metabolism in different stages of clear cell renal cell carcinoma (ccRCC). Samples of the tumor and noncancerous fragments of the same kidney were collected from patients who underwent a radical nephrectomy. The subjects were stratified according to the degree of malignancy of the tumor (n = 14 for G2, 12 for G3, and 9 for G4). The content of bioactive sphingolipids/glycosphingolipids was measured with an HPLC and HPTLC method, and the mRNA and protein expression of sphingolipid transporters and metabolizing enzymes was evaluated using real-time polymerase chain reaction (PCR) and Western blot, respectively. Compared to healthy kidney tissue, ccRCC was characterized by accumulation of sphingosine, sphingosine-1-phosphate (S1P), ceramide, dihydrosphingosine, and dihydroceramide. However, in the case of the latter two, the accumulation was limited to higher malignancy grades. In addition, compared to the healthy tissue, the content of gangliosides in the tumor was increased at the expense of globosides. We also found profound grade-dependent changes in the mRNA level of S1P-metabolizing enzymes, and spinster homolog 2. In general, their expression was much higher in G2 tumors compared to higher malignancy grades. We conclude that ccRCC is characterized by profound and multilevel alterations in sphingolipid metabolism, which to a large extent are grade-dependent. We hypothesize that dysregulation of sphingolipid metabolism contributes to the progression of ccRCC.

Exposure to Normobaric Hypoxia Combined with a Mixed Diet Contributes to Improvement in Lipid Profile in Trained Cyclists.

This study aimed to analyze the effects of live high-train low method (LH-TL) and intermittent hypoxic training (IHT) with a controlled mixed diet on lipid profile in cyclists. Thirty trained male cyclists at a national level with at least six years of training experience participated in the study. The LH-TL group was exposed to hypoxia (FiO2 = 16.5%) for 11-12 h a day and trained under normoxia for 3 weeks. In the IHT group, participants followed the IHT routine three times a week under hypoxia (FiO2 = 16.5%) at lactate threshold intensity. The control group (N) lived and trained under normoxia. The results showed that the 3-week LH-TL method significantly improved all lipid profile variables. The LH-TL group showed a significant increase in HDL-C by 9.0% and a decrease in total cholesterol (TC) by 9.2%, LDL-C by 18.2%, and triglycerides (TG) by 27.6%. There were no significant changes in lipid profiles in the IHT and N groups. ∆TG and ∆TC were significantly higher in the LH-TL group compared to the N group. In conclusion, hypoxic conditions combined with a mixed diet can induce beneficial changes in lipid profile even in highly trained athletes. The effectiveness of the hypoxic stimulus is closely related to the hypoxic training method.

Red Blood Cell 2,3-Diphosphoglycerate Decreases in Response to a 30 km Time Trial Under Hypoxia in Cyclists.

Red blood cell 2,3-diphosphoglycerate (2,3-DPG) is one of the factors of rightward-shifted oxygen dissociation curves and decrease of Hb-O2 affinity. The reduction of Hb-O2 affinity is beneficial to O2 unloading at the tissue level. In the current literature, there are no studies about the changes in 2,3-DPG level following acute exercise in moderate hypoxia in athletes. For this reason, the aim of this study was to analyze the effect of prolonged intense exercise under normoxic and hypoxic conditions on 2,3-DPG level in cyclists. Fourteen male trained cyclists performed a simulation of a 30 km time trial (TT) in normoxia and normobaric hypoxia (FiO2 = 16.5%, ~2,000 m). During the TT, the following variables were measured: power, blood oxygen saturation (SpO2), and heart rate (HR). Before and immediately after exercise, the blood level of 2,3-DPG and acid-base equilibrium were determined. The results showed that the mean SpO2 during TT in hypoxia was 8% lower than in normoxia. The reduction of SpO2 in hypoxia resulted in a decrease of average power by 9.6% (p < 0.001) and an increase in the 30 km TT completion time by 3.8% (p < 0.01) compared to normoxia. The exercise in hypoxia caused a significant (p < 0.001) decrease in 2,3-DPG level by 17.6%. After exercise in normoxia, a downward trend of 2,3-DPG level was also observed, but this effect was not statistically significant. The analysis also revealed that changes of acid-base balance were significantly larger (p < 0.05) after exercise in hypoxia than in normoxia. In conclusion, intense exercise in hypoxic conditions leads to a decrease in 2,3-DPG concentration, primarily due to exercise-induced acidosis.

Sphingosine-1-phosphate in acute exercise and training.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid found in all eukaryotic cells. Although it may function as an intracellular second messenger, most of its effects are induced extracellularly via activation of a family of five specific membrane receptors. Sphingosine-1-phosphate is enriched in plasma, where it is transported by high-density lipoprotein and albumin, as well as in erythrocytes and platelets which store and release large amounts of this sphingolipid. Sphingosine-1-phosphate regulates a host of cellular processes such as growth, proliferation, differentiation, migration, and apoptosis suppression. It was also shown to play an important role in skeletal muscle physiology and pathophysiology. In recent years, S1P metabolism in both muscle and blood was found to be modulated by exercise. In this review, we summarize the current knowledge on the effect of acute exercise and training on S1P metabolism, highlighting the role of this sphingolipid in skeletal muscle adaptation to physical effort.

Pharmacological inhibition of sphingosine-1-phosphate lyase partially reverses spatial memory impairment in streptozotocin-diabetic rats.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid with strong neuroprotective properties that is important for normal excitability and synaptic transmission in the hippocampal neurons. Considering the above, the aim of the present study was to determine whether increasing brain S1P level is able to reverse spatial memory impairment in streptozotocin-diabetic rats. The experiment was carried out on diabetic (n = 22) and nondiabetic (n = 10) male Wistar rats. Diabetes was induced by a single injection of streptozotocin. Eleven weeks later, 11 diabetic animals received injections of THI (S1P lyase inhibitor) for seven days. During the last five days of the experiment spatial reference memory acquisition and retention were tested in the Morris water maze task. The animals were then anaesthetized and samples of the hippocampus, prefrontal cortex, striatum, and cerebellum were excised. The content of S1P and related sphingolipids was measured using a HPLC method. Diabetes induced a depletion of ceramide in the hippocampus and cerebellum that was associated with impaired spatial memory and learning. Administration of THI to the diabetic animals prevented ceramide depletion in the hippocampus and cerebellum, and induced an increase in S1P content in all examined brain structures. These effects were associated with an improvement in spatial memory. We conclude that pharmacological inhibition of S1P lyase partially reverses the impairment in spatial memory induced by chronic hyperglycemia, and that this effect may be related to the prevention of ceramide depletion in the hippocampus and cerebellum, the increase in brain S1P level, or both.

Plasma concentration and expression of adipokines in epicardial and subcutaneous adipose tissue are associated with impaired left ventricular filling pattern.

BACKGROUND: Adipokines in serum derive mainly from subcutaneous and visceral adipose tissues. Epicardial adipose tissue (EAT), being a relatively small but unique fat depot, probably does not make an important contribution to systemic concentrations of adipokines. However, proximity of EAT to cardiac muscle and coronary arteries allows cells and proteins to penetrate between tissues. It is hypothesized that overexpression of proinflammatory cytokines in EAT plays an important role in pathophysiology of the heart. The aim of the study was to analyze the relationship between echocardiographic heart parameters and adipokines in plasma, epicardial, and subcutaneous fat in patients with obesity and type 2 diabetes mellitus (T2DM). Additionally, we evaluate proinflammatory properties of EAT by comparing that depot with subcutaneous adipose tissue. METHODS: The study included 55 male individuals diagnosed with coronary artery disease (CAD) who underwent planned coronary artery bypass graft. Plasma concentrations of leptin, adiponectin, resistin, visfatin, apelin, IL-6, and TNF-α, as well as their mRNA and protein expressions in EAT and subcutaneous adipose tissue (SAT) were determined. RESULTS: Obesity and diabetes were associated with increased leptin and decreased adiponectin plasma levels, higher protein expression of leptin and IL-6 in SAT, and higher visfatin protein expression in EAT. Impaired left ventricular (LV) diastolic function was associated with increased plasma concentrations of leptin, resistin, IL-6, and adiponectin, as well as with increased expressions of resistin, apelin, and adiponectin in SAT, and leptin in EAT. CONCLUSIONS: Obesity and T2DM in individuals with CAD have a limited effect on adipokines. Expression of adipokines in EAT and SAT is linked to certain heart parameters, however diastolic dysfunction of the LV is strongly associated with circulating adipokines.

Assessment of the Main Compounds of the Lipolytic System in Treadmill Running Rats: Different Response Patterns between the Right and Left Ventricle.

The aim of the present study was to investigate the time and intensity dependent effects of exercise on the heart components of the lipolytic complex. Wistar rats ran on a treadmill with the speed of 18 m/min for 30 min (M30) or 120 min (M120) or with the speed of 28 m/min for 30 min (F30). The mRNA and protein expressions of the compounds adipose triglyceride lipase (ATGL), comparative gene identification-58 (CGI-58), G0/G1 switch gene 2 (G0S2), hormone sensitive lipase (HSL) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) were examined by real-time PCR and Western blot, respectively. Lipid content of free fatty acids (FFA), diacylglycerols (DG) and triacylglycerols (TG) were estimated by gas liquid chromatography. We observed virtually no changes in the left ventricle lipid contents and only minor fluctuations in its ATGL mRNA levels. This was in contrast with its right counterpart i.e., the content of TG and DG decreased in response to both increased duration and intensity of a run. This occurred in tandem with increased mRNA expression for ATGL, CGI-58 and decreased expression of G0S2. It is concluded that exercise affects behavior of the components of the lipolytic system and the lipid content in the heart ventricles. However, changes observed in the left ventricle did not mirror those in the right one.

Muscle-Saturated Bioactive Lipids Are Increased with Aging and Influenced by High-Intensity Interval Training.

Ceramide and diacylglycerol are linked to insulin resistance in rodents, but in humans the data are inconsistent. Insulin resistance is frequently observed with aging, but the role of ceramide and diacylglycerol is not clarified. Training improves metabolic health and, therefore, we aimed to elucidate the influence of age and high-intensity interval training (HIIT) on ceramide and diacylglycerol content in muscle. Fourteen young (33 ± 1) and 22 older (63 ± 1) overweight to obese subjects performed 6 weeks HIIT three times a week. Maximal oxygen uptake and body composition were measured and muscle biopsies and fasting blood samples were obtained. Muscle ceramide and diacylglycerol were measured by gas-liquid chromatography and proteins in insulin signaling, lipid and glucose metabolism were measured by Western blotting. Content of ceramide and diacylglycerol total, saturated, C16:0 and C18:0 fatty acids and C18:1 ceramide were higher in older compared to young. HIIT reduced saturated and C18:0 ceramides, while the content of the proteins involved in glucose (GLUT4, glycogen synthase, hexokinase II, AKT) and lipid metabolism (adipose triglyceride lipase, fatty acid binding protein) were increased after HIIT. We demonstrate a higher content of saturated ceramide and diacylglycerol fatty acids in the muscle of older subjects compared to young. Moreover, the content of saturated ceramides was reduced and muscle glucose metabolism improved at protein level after HIIT. This study highlights an increased content of saturated ceramides in aging which could be speculated to influence insulin sensitivity.

The Influence of Age and Cardiorespiratory Fitness on Bioactive Lipids in Muscle.

Reduced insulin sensitivity is observed with aging and often explained by decreased physical activity. The mechanisms involved are not clarified, but bioactive lipids may play a role. We aimed to evaluate the influence of age and cardiorespiratory fitness on ceramide and diacylglycerol content in muscle and key proteins in lipid metabolism and insulin signaling. Healthy males were stratified by age into trained and untrained groups including 27 young (23.2 ± 0.3 years) and 33 aged (65.2 ± 0.6 years). Maximal oxygen uptake and body composition were measured and fasting blood samples and muscle biopsies obtained. Muscle ceramide and diacylglycerol were determined by thin-layer and gas-liquid chromatography and proteins by western blotting. We show that HOMA-IR was higher and VO2 peak lower in aged compared with young. Total, saturated, C16:0 and C18:0 ceramide content were lower in muscle from aged compared with young. Intramuscular C18:1n9 and C20:4n6 content were higher in trained versus untrained. Content of total unsaturated and C16:1n7 diacylglycerol fatty acids were higher and C24:0 lower in muscle of aged versus young. Cardiorespiratory fitness had no impact on total diacylglycerol content. In conclusion, these data argue against intramuscular ceramide or diacylglycerol accumulation as driver of age-related insulin resistance in lean individuals.

Correction: Low expression of IL-18 and IL-18 receptor in human skeletal muscle is associated with systemic and intramuscular lipid metabolism-Role of HIV lipodystrophy.

[This corrects the article DOI: 10.1371/journal.pone.0186755.].

Arteriovenous Sphingosine-1-Phosphate Differences Across Selected Organs of the Rat.

BACKGROUND/AIMS: Sphingosine-1-phosphate (S1P) is a bioactive lysosphingolipid that is found in high concentration in plasma. The majority of plasma S1P is transported bound to HDL and albumin. Although the major sources of circulating S1P have been identified, it remains obscure what is the contribution of different organs/tissues to S1P homeostasis in plasma. Answering this question was the major aim of the present study. METHODS: The experiment was performed on male Wistar rats from whom blood samples were taken from either: 1) femoral vein, right ventricle of the heart, and abdominal aorta (n=15) or 2) hepatic vein, portal vein, and abdominal aorta (n=11). Plasma was fractionated by sequential flotation ultracentrifugation and sphingolipids were quantified by a HPLC method. RESULTS: Compared to the mixed venous blood sampled from the right ventricle, total plasma and lipoprotein-depleted plasma (LPDP) concentration of S1P in the arterial blood was lower. On the other hand, the level of S1P increased across the leg both in plasma and LPDP. The concentration of S1P, sphingosine, and sphinganine in the plasma, HDL, and LPDP isolated from the blood taken from the hepatic vein was markedly higher compared to both arterial and portal blood. CONCLUSIONS: We conclude that, in contrast to HDL-bound S1P, albumin-associated S1P is very labile in the circulation. It is degraded in the pulmonary, and to a lesser extent, gastrointestinal circulation, and released across the liver and skeletal muscle. We also conclude that liver is an important source of HDL-bound S1P and circulating free sphingoid bases.

Low expression of IL-18 and IL-18 receptor in human skeletal muscle is associated with systemic and intramuscular lipid metabolism-Role of HIV lipodystrophy.

INTRODUCTION: Interleukin (IL)-18 is involved in regulation of lipid and glucose metabolism. Mice lacking whole-body IL-18 signalling are prone to develop weight gain and insulin resistance, a phenotype which is associated with impaired fat oxidation and ectopic skeletal muscle lipid deposition. IL-18 mRNA is expressed in human skeletal muscle but a role for IL-18 in muscle has not been identified. Patients with HIV-infection and lipodystrophy (LD) are characterized by lipid and glucose disturbances and increased levels of circulating IL-18. We hypothesized that skeletal muscle IL-18 and IL-18 receptor (R) expression would be altered in patients with HIV-lipodystrophy. DESIGN AND METHODS: Twenty-three HIV-infected patients with LD and 15 age-matched healthy controls were included in a cross-sectional study. Biopsies from the vastus lateralis muscle were obtained and IL-18 and IL-18R mRNA expression were measured by real-time PCR and sphingolipids (ceramides, sphingosine, sphingosine-1-Phosphate, sphinganine) were measured by HPLC. Insulin resistance was assessed by HOMA and the insulin response during an OGTT. RESULTS: Patients with HIV-LD had a 60% and 54% lower level of muscular IL-18 and IL-18R mRNA expression, respectively, compared to age-matched healthy controls. Patients with HIV-LD had a trend towards increased levels of ceramide (18.3±4.7 versus 14.8±3.0,p = 0.06) and sphingosine (0.41±0.13 versus 0.32±0.07, and lower level of sphinganine (p = 0.06). Low levels of muscle IL-18 mRNA correlated to high levels of ceramides (r = -0.31, p = 0.038) and sphingosine-1P (r = -0.29, p = 0.046) in skeletal muscle, whereas such a correlation was not found in healthy controls. Low expression of IL-18 mRNA in skeletal muscle correlated to elevated concentration of circulating triglycerides (Rp = -0.73, p<0.0001). Neither muscle expression of IL-18 mRNA or ceramide correlated to parameters of insulin resistance. CONCLUSION: IL-18 (mRNA) in skeletal muscle appears to be involved in the regulation of intramuscular lipid metabolism and hypertriglyceridemia.

Effect of metformin on bioactive lipid metabolism in insulin-resistant muscle.

Intramuscular accumulation of bioactive lipids leads to insulin resistance and type 2 diabetes (T2D). There is lack of consensus concerning which of the lipid mediators has the greatest impact on muscle insulin action in vivo Our aim was to elucidate the effects of high-fat diet (HFD) and metformin (Met) on skeletal muscle bioactive lipid accumulation and insulin resistance (IR) in rats. We employed a [U-13C]palmitate isotope tracer and mass spectrometry to measure the content and fractional synthesis rate (FSR) of intramuscular long-chain acyl-CoA (LCACoA), diacylglycerols (DAG) and ceramide (Cer). Eight weeks of HFD-induced intramuscular accumulation of LCACoA, DAG and Cer accompanied by both systemic and skeletal muscle IR. Metformin treatment improved insulin sensitivity at both systemic and muscular level by the augmentation of Akt/PKB and AS160 phosphorylation and decreased the content of DAG and Cer and their respective FSR. Principal component analysis (PCA) of lipid variables revealed that altered skeletal muscle IR was associated with lipid species containing 18-carbon acyl-chain, especially with C18:0-Cer, C18:1-Cer, 18:0/18:2-DAG and 18:2/18:2-DAG, but not palmitate-derived lipids. It is concluded that the insulin-sensitizing action of metformin in skeletal muscle is associated with decreased 18-carbon acyl-chain-derived bioactive lipids.

Training Does Not Alter Muscle Ceramide and Diacylglycerol in Offsprings of Type 2 Diabetic Patients Despite Improved Insulin Sensitivity.

Ceramide and diacylglycerol (DAG) may be involved in the early phase of insulin resistance but data are inconsistent in man. We evaluated if an increase in insulin sensitivity after endurance training was accompanied by changes in these lipids in skeletal muscle. Nineteen first-degree type 2 diabetes Offsprings (Offsprings) (age: 33.1 ± 1.4 yrs; BMI: 26.4 ± 0.4 kg/m2) and sixteen matched Controls (age: 31.3 ± 1.5 yrs; BMI: 25.3 ± 0.7 kg/m2) performed 10 weeks of endurance training three times a week at 70% of VO2max on a bicycle ergometer. Before and after the intervention a hyperinsulinemic-euglycemic clamp and VO2max test were performed and muscle biopsies obtained. Insulin sensitivity was significantly lower in Offsprings compared to control subjects (p < 0.01) but improved in both groups after 10 weeks of endurance training (Off: 17 ± 6%; Con: 12 ± 9%, p < 0.01). The content of muscle ceramide, DAG, and their subspecies were similar between groups and did not change in response to the endurance training except for an overall reduction in C22:0-Cer (p < 0.05). Finally, the intervention induced an increase in AKT protein expression (Off: 27 ± 11%; Con: 20 ± 24%, p < 0.05). This study showed no relation between insulin sensitivity and ceramide or DAG content suggesting that ceramide and DAG are not major players in the early phase of insulin resistance in human muscle.

Liver X Receptor Agonist TO901317 Prevents Diacylglycerols Accumulation in the Heart of Streptozotocin-Diabetic Rats.

BACKGROUND/AIMS: Liver X receptors (LXRα and LXRß) are ligand-activated transcription factors that regulate expression of genes involved in lipid and cholesterol metabolism. LXR expression has been identified in the heart, and enhanced LXR activity in the streptozotocin (STZ) diabetic myocardium was reported recently. The aim of this study was to investigate effect of in vivo LXR activation on myocardial lipid metabolism under conditions of STZ-induced diabetes. METHODS: Wistar rats were randomly divided into three experimental groups: non-diabetic control, treated with STZ, and treated with STZ and LXR agonist - TO901317. Diabetes was induced by a single intraperitonal injection of STZ at a dose of 55 mg/kg. LXR agonist was administrated once daily in the morning by an oral gavage at a dose of 10 mg/kg/d during the last week of the experiment. After anesthesia samples of blood and the left ventricle were taken. RESULTS: TO901317 administration increased expression of both LXR isoforms and its target genes: sterol response element binding protein 1c (SREBP-1c) and acetyl-coenzyme A carboxylase 1 (ACC1) in the heart of streptozotocin-diabetic rats. Treatment with LXR agonist had no effect on plasma lipids and glucose in the diabetic rats. Concomitantly, content of the examined lipid classes in the diabetic heart (nonesterified fatty acids, triacylglycerols, phospholipids, cholesterol esters, ceramide) was unchanged after treatment with TO901317. On the contrary, myocardial level of cholesterol and diacylglycerols (DAG) was decreased after LXR activation in diabetic rats, the change in DAG level was associated with downregulated expression of adipose triglyceride lipase (ATGL). CONCLUSION: Activation of LXRs by TO901317 protects cardiomyocytes against DAG accumulation and thus may reverse disturbances in lipid metabolism observed in streptozotocin-diabetic heart.

Reduction of ceramide de novo synthesis in solid tissues changes sphingolipid levels in rat plasma, erythrocytes and platelets.

PURPOSE: De novo sphingolipid synthesis does not occur in plasma, erythrocytes and platelets. The purpose of the study was to examine the effect of inhibition of sphingolipid synthesis in solid tissues on the level of the following bioactive sphingolipids: sphinganine, ceramide, sphingosine and sphingosine 1-phosphate in plasma, erythrocytes and platelets. MATERIAL/METHODS: The experiments were carried out on male Wistar rats. Myriocin was used to inhibit serine palmitoyltransferase activity (the enzyme catalyzes the first step of ceramide de novo synthesis) and nicotinic acid was used to reduce the concentration of plasma free fatty acids (a substrate for the de novo ceramide synthesis). The sphingolipids were quantified by means of liquid chromatography/mass spectrometry. RESULTS: Myriocin reduced the level of each compound in plasma. It reduced the level of sphinganine, sphingosine-1-phosphate and total ceramide and elevated the level of sphingosine in erythrocytes. In platelets, myriocin reduced the total level of ceramide. Nicotinic acid reduced the plasma level of sphinganine, sphingosine and total ceramide. It increased the level of sphingosine-1-phosphate in erythrocytes. In platelets, nicotinioc acid increased the level of sphinganine and sphingosine and reduced the level of sphingosine-1-phosphate and total ceramide. CONCLUSIONS: Inhibition of serine palmitoyltransferase activity in solid tissues and reduction in plasma free fatty acids concentration affects sphingolipid level in plasma, erythrocytes and platelets. The changes in erythrocytes and platelets depend both on the cell type and the sphingolipid studied and only partially follow the changes in the plasma.