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.

The implication of adipocyte ATP-binding cassette A1 and G1 transporters in metabolic complications of obesity.

Obesity is characterised by imbalance in lipid metabolism manifested by high concentrations of circulating triacylglycerols and total cholesterol as well as low high-density lipoprotein (HDL) levels. Abnormalities related to these lipids lead to metabolic complications such as type 2 diabetes, arterial hypertension and cardiovascular disease. Despite extensive research, it is still unclear why a subset of obese subjects develop metabolic syndrome, while others do not. The aim of our work was to assess total and plasma membrane expressions of cholesterol transport proteins: adipocyte ATP-binding cassette A1 (ABCA1), adipocyte ATP-binding cassette G1 (ABCG1), class B scavenger receptor (SR-BI) in visceral and subcutaneous adipose tissue of obese subjects with and without metabolic syndrome. To keep our preliminary study group uniform, we focused on women, who constitute the majority of bariatric patients. The study was performed on 34 patients: 24 morbidly obese women subjected to bariatric surgery, half of whom had metabolic syndrome; and 10 lean subjects undergoing elective laparoscopic cholecystectomy. Total and plasma membrane expressions of cholesterol transport proteins (SR-BI, ABCA1 and ABCG1) were assessed in samples of both visceral and subcutaneous adipose and analysed in relation to other clinical and laboratory parameters. We demonstrated lower plasma membrane expressions of ABCG1 in visceral adipose tissue of obese patients with metabolic syndrome as compared to lean ones. In addition, total ABCG1 expressions in both types of adipose tissue were lower in morbidly obese patients with metabolic syndrome compared to those without metabolic syndrome. Plasma membrane ABCA1 expressions in visceral adipose tissue were lower in the group of morbidly obese patients without metabolic syndrome, compared to lean patients. We did not find any significant differences in SR-BI expressions. Because of ABCG1 is responsible for cholesterol efflux to HDL, reduced plasma membrane expression of ABCG1 in VAT of morbidly obese women with metabolic syndrome may leads to a significantly decreased concentration of HDL in serum. This may be also confirmed by high positive correlation between both parameters.

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.

Fiber specific changes in sphingolipid metabolism in skeletal muscles of hyperthyroid rats.

Thyroid hormones (T3, T4) are well known modulators of different cellular signals including the sphingomyelin pathway. However, studies regarding downstream effects of T3 on sphingolipid metabolism in skeletal muscle are scarce. In the present work we sought to investigate the effects of hyperthyroidism on the activity of the key enzymes of ceramide metabolism as well as the content of fundamental sphingolipids. Based on fiber/metabolic differences, we chose three different skeletal muscles, with diverse fiber compositions: soleus (slow-twitch oxidative), red (fast-twitch oxidative-glycolytic) and white (fast-twitch glycolytic) section of gastrocnemius. We demonstrated that T3 induced accumulation of sphinganine, ceramide, sphingosine, as well as sphingomyelin, mostly in soleus and in red, but not white section of gastrocnemius. Concomitantly, the activity of serine palmitoyltransferase and acid/neutral ceramidase was increased in more oxidative muscles. In conclusion, hyperthyroidism induced fiber specific changes in the content of sphingolipids that were relatively more related to de novo synthesis of ceramide rather than to its generation via hydrolysis of sphingomyelin.

Fatty acid transporters involved in the palmitate and oleate induced insulin resistance in primary rat hepatocytes.

AIMS: To determine the presence and possible involvement of FAT/CD36, FABPpm and FATP-2, transporters in (i) fatty acids movement across plasma membrane and (ii) an induction of insulin resistance by palmitic (PA) and oleic (OA) fatty acids in primary hepatocytes. METHODS: Primary hepatocytes were treated with either PA and OA or combination of activators (AICAR, Insulin) or inhibitors (SSO, phloretin) of FA transport. Expression of FA and glucose transporters as well as insulin signalling proteins was determined using Western blot analyses. Palmitate and glucose transport was measured using radioactive isotopes. Intracellular lipid content [ceramide, diacylglycerols (DG) and triacylglycerols] and FA composition were estimated by GLC. RESULTS: In primary hepatocytes, adding phloretin diminished insulin, and AICAR stimulated palmitate transport. Both PA and OA fatty acids induced the protein expression of FAT/CD36 and FATP-2 with concomitant: (i) reduction in GLUT-2 protein content, (ii) inhibition of insulin-stimulated glucose uptake, (iii) reduction in insulin-stimulated activation of AKT and GSK, (iv) accumulation of either DG (PA and OA) or ceramide (only PA). CONCLUSIONS: FA transport into hepatocytes is, at least in part, protein-mediated process, and both PA and OA induce the protein expression of FAT/CD36 and FATP-2. Both saturated (PA) and unsaturated (OA) fatty acids induce insulin resistance in primary hepatocytes, associated with the accumulation of DG and/or ceramide.

Effects of hyperthyroidism on lipid content and composition in oxidative and glycolytic muscles in rats.

Triiodothyronine (T(3)) can influence lipid metabolism via multiple mechanisms, which generally result in an increase of fatty acids (FAs) oxidation. Consequently, we hypothesize that hyperthyroidism may influence intramuscular lipids accumulation. This increased intramuscular lipid turn-over is possibly accompanied by an increase in fatty acid transporters expression (FAT/CD36, FABPpm, FATP-1,4). In the present study we examined the lipid content and fatty acid saturation status of free fatty acids (FFA), triacylglycerols (TAG), diacylglycerols (DAG) and phospholipids (PL) in skeletal muscle of hyperthyroid rats (n=8). We measured also fatty acid transporters as well as AMP-activated protein kinase (pAMPK/AMPK), peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), acetyl-CoA carboxylase (pACC/ACC), carnitine palmitoyltransferase I (CPT I) and citrate synthase (CS) protein expression in these muscles. In vivo T3 administration, decreased the content of FFA, particularly in the red gastrocnemius and the TAG fraction, in both the red and white portions of the gastrocnemius muscle. Concomitantly, saturated/unsaturated fatty acids (SFA/UFA) ratio was also decreased, but only in the FFA fraction, irrespectively of muscle's fiber composition. In contrast, T(3) treatment had no effect on the lipid content and saturation status in PL fraction. Triiodothyronine induced also modest activation of AMPK/ACC axis with subsequent increased expression of mitochondrial proteins: CPT I and CS. This was accompanied by increased content of FAT/CD36, but only in the red part of gastrocnemius muscle. These findings support the conclusion that hyperthyroidism increases lipid metabolism, especially in skeletal muscles with high capacity for fatty acid oxidation.