Effect of acute exercise on mRNA and protein expression of main components of the lipolytic complex in different skeletal muscle types in the rat.

Adipose triglyceride lipase (ATGL) hydrolyses the first bond of triacylglycerols. The activity of the enzyme is elevated by comparative gene identification 58 (CGI-58), and reduced by G0/G1 switch gene 2 (G0S2) protein. There are no data on the effect of acute exercise on the behavior of particular components of the lipolytic complex in different skeletal muscle types, therefore, the aim of the present study was to examine that topic. The experiments were carried out on four groups of male Wistar rats: 1) control 2) rats running on a treadmill at the speed of 18 m/min for 30 min, 3) at the speed of 18 m/min for 120 min, 4) for 30 min at the speed of 28 m/min. We found that each exercise bout induced numerous changes in the expression of mRNA and protein ATGL, hormone-sensitive lipase, CGI-58 and G0S2 in the investigated muscles. These changes, depended to a large extent on a muscle type. In general, the strongest pro-lipolytic response was observed in the soleus, followed by the red section of the gastrocnemius (RG). On the other hand, in the white section of the gastrocnemius protein expression of the components of the lipolytic complex was reduced in response to exercise. These changes were not accompanied by alterations in muscle triacylglycerol content, with the exception of a reduction observed in the RG following 2-hour run. We conclude that a single bout of exercise induces significant effect on the expression of components of the lipolytic complex in skeletal muscle, and that the magnitude of this effect depends on muscle oxidative capacity, as well as the duration and intensity of exercise.

Endurance training selectively increases high-density lipoprotein-bound sphingosine-1-phosphate in the plasma.

Sphingosine-1-phosphate (S1P) is a bioactive lysosphingolipid that is found in relatively high concentration in human plasma. Erythrocytes, endothelial cells, and activated platelets are the main sources of circulating S1P. The majority of plasma S1P is transported bound to high-density lipoprotein (HDL) and albumin. In recent years, HDL-bound S1P attracted much attention due to its cardioprotective and anti-atherogenic properties. We have previously found that endurance-trained athletes are characterized by higher plasma S1P concentration compared to untrained individuals. This finding prompted us to examine the effect of endurance training on S1P metabolism in blood. Thirteen healthy, untrained, male subjects completed an 8-week training program on a rowing ergometer. Three days before the first, and 3 days after the last training session, blood samples were drawn from an antecubital vein. We found that total plasma S1P concentration was increased after the training. Further analysis of different plasma fractions showed that the training selectively elevated HDL-bound S1P. This effect was associated with activation of sphingosine kinase in erythrocytes and platelets and enhanced S1P release from red blood cells. We postulate that increase in HDL-bound S1P level is one of the mechanisms underlying beneficial effects of regular physical activity on cardiovascular diseases.

Effect of tachycardia on mRNA andf protein expression of the principal components of the lipolytic system in the rat's heart ventricles.

There is a convincing piece of evidence showing that most of free fatty acids (FFA) entering cardiomyocytes are first esterified into triacylglycerols (TG) before being oxidized or used for synthesis of complex lipids. The enzyme adipose triglyceride lipase (ATGL) initiates lipolysis of TG by hydrolyzing the first ester bond of the compound. As a result, free fatty acid and diacylglycerol (DG) are released in that process. Finally, DG may be further hydrolyzed by the enzyme hormone sensitive lipase (HSL). The aim of the present study was to examine effect of tachycardia on mRNA and protein expression of ATGL, CGI-58 (an activator of ATGL), G0S2 (an inhibitor of ATGL) and HSL in the left and right ventricle of the rat. The experiments were carried out on male Wistar rats, 240 - 260 grams of body weight. After anesthesia, two electrodes were inserted in the right jugular vein and connected to SC-04 stimulator. The rats were randomly allocated into one of the three groups, namely: control, 30 min and 60 min of the heart stimulation at the rate of 600 times/min. The expressions of ATGL, CGI-58, G0S2 and HSL were evaluated at both gene and protein levels using real-time PCR and Western Blot analysis, respectively. Both 30 and 60 min stimulation reduced ATGL, HSL, CGI-58 and G0S2 mRNA content in the left ventricle. The stimulation had only insignificant impact on ATGL, HSL and CGI-58 transcript levels in the right ventricle. Interestingly, Western Blot analysis showed that the stimulation, regardless of the time, reduced the ATGL and G0S2 protein expression, but did not change the CGI-58 and HSL expression in the left ventricle. Furthermore, in the right ventricle, the protein expressions of ATGL, HSL and G0S2 decreased after stimulation. In conclusion, the obtained results clearly show that tachycardia affects both mRNA and protein expression of particular compounds of the TG lipolytic system in the heart ventricles. Additionally, there are marked differences in the expressions of the examined compounds between the ventricles.

Effect of tachycardia on lipid metabolism and expression of fatty acid transporters in heart ventricles of the rat.

Tachycardia increases oxidation of the plasma-borne long chain fatty acids in the heart. The aim of the present study was to examine effect of tachycardia on: 1) the total level of free fatty acids, diacylglycerols, triacylglycerols and phospholipids in both heart ventricles; 2) (14)C-palmitate incorporation in the lipid fractions; 3) expression of fatty acid and glucose transporters in the ventricles. Tachycardia was induced in anesthetized rats by electrical atrial pacing at the rate of 600/min. Samples of the left (LV) and right (RV) ventricle were taken after 30 and 60 min pacing. The level free fatty acids, diacylglycerols, triacylglycerols and phospholipids was determined by means of gas-liquid chromatography and (14)C-palmitate incorporation by liquid scintillation counting, respectively. Expression of fatty acid- and glucose-transporters was determined using Western blot technique. In LV, 30min pacing increased the content of diacylglycerols whereas the content of other lipids remained stable. After 60 min of pacing the levels of the examined lipid fractions did not differ from the respective control values. In RV, the content of diacylglycerols and triacylglycerols was reduced both after 30 and 60 min pacing. Tachycardia also affected incorporation of (14)C-palmitate in lipid fractions of goth ventricles. 30 min pacing up-regulated plasmalemmal expression of FAT/CD36 (fatty acid translocase) in both ventricles and reduced its microsomal expression in LV. After 60 min pacing they did not differ from the respective control values. Plasmalemmal expression of FATP-1 (fatty acid transport protein 1) increased and its microsomal expression decreased in RV after 30 min pacing. After 60 min pacing the plasmalemmal FATP-1 expression remained elevated whereas the microsomal expression did not differ from the control value. Pacing did not affect or expression of FABPpm (plasma membrane associated fatty acid binding protein) in either plasma membranes and microsomal compartments. Thirty min pacing increased plasmalemmal and reduced microsomal expression of GLUT-4 (glucotransporter 4) in both ventricles. It increased plasmalemmal expression of GLUT-1 (glucotransporter 1) in RV. It returned to normal after 60 min pacing. It is concluded that tachycardia induces numerous changes in metabolism of myocardial lipids as well as expression of fatty acid and glucose transporters in both heart ventricles.

Liver X receptor agonist T0901317 enhanced peroxisome proliferator-activated receptor-delta expression and fatty acid oxidation in rat skeletal muscle.

Liver X receptors (LXR) have been characterized as key transcriptional regulators of hepatic lipid and carbohydrate metabolism. LXR are expressed also in skeletal muscle, however, their role in this tissue is poorly investigated and the vast majority of available data comes from studies on cultured myotubes. Therefore, we aimed to examine effects of in vivo LXR activation on muscle lipid metabolism. The experiments were performed on male Wistar rats fed on a standard rodent chow. The animals were divided into two groups (n=10) receiving either LXR activator (T0901317, 10 mg/kg/day) or vehicle for one week. Samples of the soleus as well as red and white sections of the gastrocnemius muscle were excised. T0901317 increased muscle expression of peroxisome proliferator-activated receptor-δ and its target genes involved in fatty acid uptake and oxidation. In addition, LXR agonist enhanced palmitate oxidation (by 55%) in isolated soleus muscle. However, palmitate incorporation into triacylglycerol was decreased (by 38%), which was associated with reduced diacylglycerol acyltransferase expression (by 66%). Despite markedly increased plasma lipid concentration upon T0901317 treatment, muscle triacylglycerol level was elevated only in the red section of the gastrocnemius muscle. We conclude that T0901317 enhances muscle fatty acid oxidation, which prevents overt accumulation of intramuscular lipids that could be expected considering T0901317-induced hyperlipidemia.

Autophagy-lysosomal pathway is involved in lipid degradation in rat liver.

We present data supporting the hypothesis that the lysosomal-autophagy pathway is involved in the degradation of intracellular triacylglycerols in the liver. In primary hepatocytes cultivated in the absence of exogenous fatty acids (FFA), both inhibition of autophagy flux (asparagine) or lysosomal activity (chloroquine) decreased secretion of VLDL (very low density lipoproteins) and formation of FFA oxidative products while the stimulation of autophagy by rapamycine increased some of these parameters. Effect of rapamycine was completely abolished by inactivation of lysosomes. Similarly, when autophagic activity was influenced by cultivating the hepatocytes in "starving" (amino-acid poor medium) or "fed" (serum-supplemented medium) conditions, VLDL secretion and FFA oxidation mirrored the changes in autophagy being higher in starvation and lower in fed state. Autophagy inhibition as well as lysosomal inactivation depressed FFA and DAG (diacylglycerol) formation in liver slices in vitro. In vivo, intensity of lysosomal lipid degradation depends on the formation of autophagolysosomes, i.e. structures bringing the substrate for degradation and lysosomal enzymes into contact. We demonstrated that lysosomal lipase (LAL) activity in liver autophagolysosomal fraction was up-regulated in fasting and down-regulated in fed state together with the increased translocation of LAL and LAMP2 proteins from lysosomal pool to this fraction. Changes in autophagy intensity (LC3-II/LC3-I ratio) followed a similar pattern.

Ceramide metabolism is affected by obesity and diabetes in human adipose tissue.

Ceramide is involved in development of insulin resistance. However, there are no data on ceramide metabolism in human adipose tissue. The aim of our study was to examine sphingolipid metabolism in fat tissue from obese nondiabetic (n = 11), obese diabetic (n = 11), and lean nondiabetic (n = 8) subjects. The content of ceramide (Cer), dihydroceramide (dhCer), sphingosine (SPH), sphinganine (SPA), sphingosine-1-phosphate (S1P; pmol/mg of protein), the expression (mRNA) and activity of key enzymes responsible for Cer metabolism: serine palmitoyltransferase (SPT), neutral and acidic sphingomyelinase (nSMase and aSMase, respectively), and neutral and acidic ceramidase (nCDase and aCDase, respectively) were examined in human adipose tissue. The contents of SPA and Cer were significantly lower whereas the content of dhCer was higher in both obese groups than the respective values in the lean subjects. The expression of examined enzymes was elevated in both obese groups. The SPT and CDases activity increased whereas aSMase activity deceased in both obese groups. We have found correlation between adipose tissue Cer content and plasma adiponectin concentration (r = 0.69, P < 0.001) and negative correlation between total Cer content and HOMA-IR index (homeostasis model of insulin resistance) (r = -0.67, P < 0.001). We have found that both obesity and diabetes affected pathways of sphingolipid metabolism in the adipose tissue.

LXR activation prevents exhaustive exercise-induced hypoglycaemia and spares muscle glycogen but does not enhance running endurance in untrained rats.

AIM: Liver X receptors (LXRs) are ligand-activated transcription factors that play an important role in regulation of hepatic lipid and carbohydrate metabolism. However, to date there is very few information on the role of LXRs in skeletal muscle. Moreover, it remains obscure whether LXR activation affects physical endurance. Therefore, we aimed to examine effects of selective LXR activator--T0901317--on running endurance and skeletal muscle exercise metabolism in rats. METHODS: The animals were assigned to two groups (n=20) receiving either vehicle or T0901317 (10 mg kg(-1) day(-1) ) for 1 week. One day after the final administration, half of the rats in each group were exercised until exhaustion on the electrically driven treadmill. All animals were then anaesthetized and samples of the soleus, red and white sections of the gastrocnemius muscle, epididymal fat pad and liver were excised. RESULTS: We found that LXR activation prevented exhaustive exercise-induced hypoglycaemia. T0901317 also shifted substrate utilization in working muscles in favour of fatty acids as indicated by its glycogen sparing effect, enhanced consumption of intramuscular triacylglycerol and upregulation of genes promoting fatty acid oxidation and suppressing carbohydrate oxidation. However, running time to exhaustion was not improved. CONCLUSION: We conclude that LXR activation increases fatty acid utilization during exercise which, however, does not translate into measurable enhancement of exercise endurance.

Effects of streptozotocin-induced diabetes and elevation of plasma FFA on ceramide metabolism in rat skeletal muscle.

Ceramide is likely to mediate in induction of insulin resistance. The aim of the present study was to examine the effect of streptozotocin-diabetes and treatment with heparin on ceramide metabolism in skeletal muscles. The experiments were performed on Wistar rats divided into three groups: 1) control, 2) treated with streptozotocin, and 3) treated with heparin. Assays were carried out on three types of muscle: slow-twitch oxidative (soleus), fast-twitch oxidative-glycolytic, and fast-twitch glycolytic (red and white section of the gastrocnemius, respectively). The activity of serine palmitoyltransferase (SPT), neutral and acid sphingomyelinase (nSMase and aSMase), and neutral and alkaline ceramidase (nCDase and alCDase) was examined. The content of ceramide, sphinganine, sphingosine, and sphingosine-1-phosphate was also measured. Both streptozotocin-diabetes and treatment with heparin increased the activity of SPT in each type of muscle. Heparin inhibits the activity of aSMase and concomitantly induces the activity of nSMase in each studied muscle. Streptozotocin decreased aSMase activity in each muscle and increased nSMase activity in the soleus and red section of the gastrocnemius. Heparin induced, whereas streptozotocin inhibited the activity of n-CDase in the soleus and the red section of the gastrocnemius. Heparin increased the activity of alCDase in the red gastrocnemius. In the soleus and the white gastrocnemius the activity of alCDase decreased. Streptozotocin significantly increased the content of ceramide in each muscle studied and heparin did it only in the soleus. It is concluded that insulin deficiency is accompanied by alterations in ceramide metabolism in skeletal muscles. Increased concentration of the plasma free fatty acids may mediate certain effects of insulin deficiency.

Effect of exercise duration on ceramide metabolism in the rat heart.

AIM: We aimed at gaining more insight into the mechanisms underlying exercise-induced alterations in myocardial ceramide (CER) content by employing physical activity of various durations and examining all key pathways of CER metabolism. METHODS: The experiments were carried out on male Wistar rats divided into four groups (n = 6 in each case): control, exercised for 30 and 90 min and until exhaustion on the electrically driven treadmill moving with a speed of 1200 m h(-1) and set at +10 degrees incline. The animals were anaesthetized and samples of the heart's left ventricle were excised. RESULTS: Thirty-minute exercise decreased the level of CER in the heart by 15%. However, after 90 min of running it returned to the baseline and at the point of exhaustion it exceeded that of the control animals by 26%. The initial reduction in the content of CER was probably a result of its augmented degradation, as a concomitant elevation in the activity of acid ceramidase and the level of sphingosine was observed. The transition from reduction in CER content after 30 min of exercise to its accumulation at the point of exhaustion was a consequence of gradual reduction in the activity of acid ceramidase and simultaneous increase in the rate of de novo CER synthesis, as evidenced by progressive activation of serine palmitoyltransferase and accumulation of sphinganine. CONCLUSION: We conclude that the effect of physical effort on myocardial CER content and metabolism depends to a large extent on exercise duration.

Increased skeletal muscle ceramide level in men at risk of developing type 2 diabetes.

AIMS/HYPOTHESIS: Intramyocellular lipids, including ceramide, a second messenger in the sphingomyelin signalling pathway, might contribute to the development of insulin resistance. The aim of our study was to assess parameters of the skeletal muscle sphingomyelin signalling pathway in men at risk of developing type 2 diabetes. METHODS: We studied 12 lean (BMI < 25 kg/m(2)) men without a family history of diabetes (control group), 12 lean male offspring of type 2 diabetic patients, and 21 men with overweight or obesity comprising 12 with NGT (obese-NGT) and nine with IGT (obese-IGT). A euglycaemic-hyperinsulinaemic clamp and a biopsy of vastus lateralis muscle were performed. Ceramide, sphingomyelin, sphinganine and sphingosine levels and sphingomyelinase and ceramidase activities were measured in muscle. Muscle diacylglycerol and triacylglycerol levels were estimated in a subgroup of 27 men (comprising men from all the above groups). RESULTS: Compared with the control group, the lean offspring of diabetic patients and the men with overweight or obesity showed lower insulin sensitivity (all p < 0.005) and a greater muscle ceramide level (all p < 0.01). The obese-IGT group had lower insulin sensitivity (p = 0.0018) and higher muscle ceramide (p = 0.0022) than the obese-NGT group. There was lower muscle sphingosine level and alkaline ceramidase activity in offspring of diabetic patients (p = 0.038 and p = 0.031, respectively) and higher sphinganine level in the obese-NGT (p = 0.049) and obese-IGT (p = 0.002) groups than in the control group. Muscle sphingomyelin was lower (p = 0.0028) and neutral sphingomyelinase activity was higher (p = 0.00079) in the obese-IGT than in the obese-NGT group. Muscle ceramide was related to insulin sensitivity independently of other muscle lipid fractions. CONCLUSIONS/INTERPRETATIONS: Ceramide accumulates in muscle of men at risk of developing type 2 diabetes.

PPARalpha agonist induces the accumulation of ceramide in the heart of rats fed high-fat diet.

It was shown that high-fat feeding of mice with cardiac-specific overexpression of peroxisome proliferator-activated receptor (PPAR) alpha but not wild type animals leads to the accumulation of ceramide (an important mediator of lipotoxicity) in the heart [Finck et al. 2003 Proc Natl Acad Sci USA]. To investigate the mechanism of this phenomenon we examined the effects of PPARalpha activation on ceramide metabolism in the myocardium. Male Wistar rats were fed either a standard chow or a high-fat diet. Each group was divided into two subgroups: control and treated with selective PPARalpha activator - WY-14643. In the rats fed on the standard diet WY-14643 did not affect the myocardial content of sphingomyelin and ceramide but reduced the content of sphinganine and sphingosine. It also inhibited the activity of neutral sphingomyelinase and increased the activity of acid sphingomyelinase, whereas the activity of ceramidases and serine palmitoyltransferase (SPT) remained stable. High-fat diet itself did not affect the content of the examined sphingolipids. However, it reduced the activity of sphingomyelinases and ceramidases having no effect on the activity of SPT. Administration of WY-14643 to this group significantly increased the content of myocardial free palmitate, ceramide, sphingomyelin and the activity of SPT. Our results demonstrated that PPARalpha activation modulates myocardial ceramide metabolism and leads to the accumulation of ceramide in the heart of the high-fat fed rats due to its increased synthesis de novo.

Effects of pioglitazone and high-fat diet on ceramide metabolism in rat skeletal muscles.

Ceramide is involved in the pathogenesis of insulin resistance in skeletal muscles of humans and rodents. However, there are conflicting reports in the literature on the effect of thiazolidinediones (a new class of insulin sensitizing drugs) on skeletal muscle ceramide content. Therefore, the aim of our study was to examine the effect of pioglitazone on the level of ceramide and its metabolites and on the activity of the key enzymes of ceramide metabolism in different skeletal muscle types of the rat. The experiments were carried out on rats fed either a standard chow or a high-fat diet for 21 days. Each group was divided into two subgroups: control and treated with pioglitazone for 14 days. High-fat diet increased the content of ceramide in the soleus and in the red section of the gastrocnemius, but not in the white section of the latter. The activity of neutral Mg(2+)-dependent sphingomyelinase and acid sphingomyelinase was simultaneously reduced in all examined muscles. Administration of pioglitazone decreased ceramide level in the soleus and in the red section of the gastrocnemius in rats fed either diet. This effect could not be attributed to decreased rate of ceramide formation from sphingomyelin or to its augmented deacylation to sphingosine. Pioglitazone treatment reduced the concentration of plasma free fatty acids in rats fed on either diet. Therefore, we conclude that the drug decreased the muscle content of ceramide by reducing its de novo synthesis. The results of our study indicate that reduction in ceramide level may be one of the mechanisms by which pioglitazone improves skeletal muscle insulin sensitivity.

Necrotizing enterocolitis: feeding in endemic and epidemic periods.

Aggressive feeding practices are thought to increase the incidence of necrotizing enterocolitis (NEC). Detailed feeding histories of the 39 cases occurring between January 1, 1984 and May 31, 1985 were compared with matched controls. The study period included a cluster (epidemic) of 11 cases diagnosed within 1 month. Data were analyzed collectively and separately for endemic and epidemic cases. Collectively, cases had greater average daily intake volume, maximum daily intake volume, intake volume on the day prior to diagnosis, and maximum daily caloric intake (all p less than 0.05) than controls. The only recorded parameter that differed in endemic cases vs controls was intake the day prior to diagnosis. In contrast, epidemic cases were fed significantly more volume, more calories, and faster than controls: average intake volume (62.5 +/- 27.2 vs 37.4 +/- 18.0 ml/kg/day); maximum intake volume (118.5 +/- 33.5 vs 76.4 +/- 38.8 ml/kg/day); intake day prior to diagnosis (109.8 +/- 30.9 vs 63.8 +/- 43.1 ml/kg/day); maximum daily increment (42.6 +/- 16.7 vs 26.7 +/- 16.4 ml/kg); maximum caloric intake (126.1 +/- 44.6 vs 77.3 +/- 50.0) (all p less than 0.01). Five of the feeding parameters were significantly less for the epidemic controls than the endemic controls, suggesting a general slowing of feeding during the NEC epidemic. In summary, the data suggest feeding patterns may have an impact on NEC especially during epidemic periods.