Effects of activation of endocannabinoid system on myocardial metabolism.

Endocannabinoids exert their effect on the regulation of energy homeostasis via activation of specific receptors. They control food intake, secretion of insulin, lipids and glucose metabolism, lipid storage. Long chain fatty acids are the main myocardial energy substrate. However, the heart exerts enormous metabolic flexibility emphasized by its ability to utilzation not only fatty acids, but also glucose, lactate and ketone bodies. Endocannabinoids can directly act on the cardiomyocytes through the CB1 and CB2 receptors present in cardiomyocytes. It appears that direct activation of CB1 receptors promotes increased lipogenesis, pericardial steatosis and bioelectrical dysfunction of the heart. In contrast, stimulation of CB2 receptors exhibits cardioprotective properties, helping to maintain appropriate amount of ATP in cardiomyocytes. Furthermore, the effects of endocannabinoids at both the central nervous system and peripheral tissues, such as liver, pancreas, or adipose tissue, resulting indirectly in plasma availability of energy substrates and affects myocardial metabolism. To date, there is little evidence that describes effects of activation of the endocannabinoid system in the cardiovascular system under physiological conditions. In the present paper the impact of metabolic diseases, i. e. obesity and diabetes, as well as the cardiovascular diseases - hypertension, myocardial ischemia and myocardial infarction on the deregulation of the endocannabinoid system and its effect on the metabolism are described.

Protective role of cannabinoid CB1 receptors and vascular effects of chronic administration of FAAH inhibitor URB597 in DOCA-salt hypertensive rats.

AIMS: This study examined whether the fall in blood pressure (BP) induced by the chronic inhibition of fatty acid amide hydrolase (FAAH) by URB597 in deoxycorticosterone acetate (DOCA-salt) hypertensive rats correlates with endocannabinoid-mediated vascular changes. MAIN METHODS: Functional studies were performed in isolated endothelium-intact aortas and small mesenteric arteries (sMAs) using organ bath technique and wire myography, respectively. KEY FINDINGS: In the DOCA-salt rats, methanandamide-stimulated relaxation was enhanced in sMAs or diminished in aortas. Its vasorelaxant effect in sMAs was sensitive to the antagonist of the Transient Receptor Potential Vanilloid type 1 (TRPV1), capsazepine, in normo- and hypertensive animals and to the antagonist of the cannabinoid CB1 receptors, AM6545, only in DOCA-salt rats. Cannabinoid CB1 receptors were up-regulated merely in DOCA-salt sMAs. URB597 decreased elevated BP in DOCA-salt rats, medial hypertrophy in DOCA-salt aortas. In sMAs it reduced FAAH expression and restored the augmented phenylephrine-induced contraction in the DOCA-salt rats to the level obtained in normotensive controls. In normotensive rats it diminished endothelium-dependent relaxation and increased phenylephrine-induced contraction. SIGNIFICANCE: The study showed the protective role of cannabinoid CB1 receptors in DOCA-salt sMAs. Reduction in BP after chronic administration of the FAAH inhibitor URB597 in DOCA-salt hypertensive rats only partially correlates with structural and functional changes in conductance and resistance vessels, respectively. Caution should be taken in studying cannabinoids and FAAH inhibitors as potential therapeutics, because of their vessel- and model-specific activities, and side effects connected with off-target response and activation of alternative pathways of anandamide metabolism.

Myocardial lipid profiling during time course of high fat diet and its relationship to the expression of fatty acid transporters.

BACKGROUND/AIMS: It is well documented that increased fatty acids (FA) supply causes lipid accumulation and insulin resistance in skeletal muscles. Whether the same mechanism is present in the heart is still unclear. Therefore, the goal of our study was to determine the content of specific myocardial lipid fractions during feeding rats a high fat diet (HFD) for 5 weeks. Moreover, the relation between changes in myocardial lipid content, whole body insulin resistance and the expression of fatty acid transporters in each week of HFD was established. METHODS: Gas liquid chromatography and high performance liquid chromatography were used to determine the content of lipid fractions in the left ventricle. Expression of selected proteins was estimated by Western blot technique. All measurements were made after each week of HFD. RESULTS: As expected, lipid profile in myocardium was altered by HFD in different weeks of the study with the most intense changes in triacylglycerols, long chain fatty acid-CoA and ceramide. Furthermore, there was a significant elevation of plasmalemmal (the 4th and the 5th week) and mitochondrial expression (from the 3rd to the 5th week) of fatty acid translocase. CONCLUSION: High fat diet affects myocardial lipid profile in each week of its duration and causes alternations in FA metabolism in cardiomyocytes.

Extremely rapid increase in fatty acid transport and intramyocellular lipid accumulation but markedly delayed insulin resistance after high fat feeding in rats.

AIMS/HYPOTHESIS: The mechanisms for diet-induced intramyocellular lipid accumulation and its association with insulin resistance remain contentious. In a detailed time-course study in rats, we examined whether a high-fat diet increased intramyocellular lipid accumulation via alterations in fatty acid translocase (FAT/CD36)-mediated fatty acid transport, selected enzymes and/or fatty acid oxidation, and whether intramyocellular lipid accretion coincided with the onset of insulin resistance. METHODS: We measured, daily (on days 1-7) and/or weekly (for 6 weeks), the diet-induced changes in circulating substrates, insulin, sarcolemmal substrate transporters and transport, selected enzymes, intramyocellular lipids, mitochondrial fatty acid oxidation and basal and insulin-stimulated sarcolemmal GLUT4 and glucose transport. We also examined whether upregulating fatty acid oxidation improved glucose transport in insulin-resistant muscles. Finally, in Cd36-knockout mice, we examined the role of FAT/CD36 in intramyocellular lipid accumulation, insulin sensitivity and diet-induced glucose intolerance. RESULTS: Within 2-3 days, diet-induced increases occurred in insulin, sarcolemmal FAT/CD36 (but not fatty acid binding protein [FABPpm] or fatty acid transporter [FATP]1 or 4), fatty acid transport and intramyocellular triacylglycerol, diacylglycerol and ceramide, independent of enzymatic changes or muscle fatty acid oxidation. Diet-induced increases in mitochondria and mitochondrial fatty acid oxidation and impairments in insulin-stimulated glucose transport and GLUT4 translocation occurred much later (≥21 days). FAT/CD36 ablation impaired insulin-stimulated fatty acid transport and lipid accumulation, improved insulin sensitivity and prevented diet-induced glucose intolerance. Increasing fatty acid oxidation in insulin-resistant muscles improved glucose transport. CONCLUSIONS/INTERPRETATIONS: High-fat feeding rapidly increases intramyocellular lipids (in 2-3 days) via insulin-mediated upregulation of sarcolemmal FAT/CD36 and fatty acid transport. The 16-19 day delay in the onset of insulin resistance suggests that additional mechanisms besides intramyocellular lipids contribute to this pathology.

New evidence for the role of ceramide in the development of hepatic insulin resistance.

AIM: There are few and contradictory data on the role of excessive accumulation of intracellular sphingolipids, particularly ceramides, in the development of hepatic insulin resistance. In our study we assessed accumulated sphingolipid fractions and clarify the mechanisms of hepatic insulin resistance development as well as involvement of fatty acid and ceramide transporters in this process. METHODS: In culture of primary rat hepatocytes, exposed to high concentration of palmitic acid (0.75 mM) during short and prolonged incubation, high performance liquid chromatography was used to assess intra- and extracellular sphingolipid fractions content. Degree of palmitate-induced insulin resistance was estimated by measuring changes in phosphorylation of insulin pathway proteins by western blotting as well as changes in expression of different type of transporters. RESULTS: In our study short and prolonged exposure of primary hepatocytes to palmitic acid resulted in increased intracellular accumulation of ceramide which inhibited insulin signaling pathway. We observed a significant increase in the expression of fatty-acid transport protein (FATP2) and ceramide transfer protein (CERT) what is consistent with enhanced intracellular ceramide content. The content of extracellular ceramide was increased nearly threefold after short and twofold after long incubation period. Expression of microsomal triglyceride transfer protein (MTP) and ATP-binding cassette transporter (ABCA1) was increased significantly mainly after short palmitate incubation. CONCLUSION: Our data showed that increase in intarcellular ceramide content contributes to the development of hepatic insulin resistance. We suggest pivotal role of transporters in facilitating fatty acid influx (FATP2), accumulation of ceramides (CERT) and export to the media (MTP and ABCA1).

Insulin-sensitizing effect of LXR agonist T0901317 in high-fat fed rats is associated with restored muscle GLUT4 expression and insulin-stimulated AS160 phosphorylation.

BACKGROUND/AIM: Liver X receptors (LXRs) are ligand-activated transcription factors that were shown to stimulate hepatic lipogenesis leading to liver steatosis and hypertriglyceridemia. Despite their pro-lipogenic action, LXR activators normalize glycemia and improve insulin sensitivity in rodent models of type 2 diabetes. Antidiabetic action of LXR agonists is thought to result from suppression of hepatic gluconeogenesis. However, it remains unclear whether LXR activation affects muscle insulin sensitivity. In the present study we attempted to answer this question. METHODS: The experiments were performed on male Wistar rats fed for 5 weeks on either standard chow or high fat diet. The latter group was further divided into two subgroups receiving either selective LXR agonist - T0901317 (10mg/kg/d) or vehicle during the last week of the experiment. All animals were then anaesthetized and samples of the soleus as well as red and white sections of the gastrocnemius muscle were excised. RESULTS: As expected, administration of T0901317 to high-fat fed rats augmented diet-induced hyperlipidemia. Nevertheless, it also normalized glucose tolerance and improved insulin-stimulated glucose uptake in isolated soleus muscle. In addition, LXR agonist completely restored glucose transporter 4 expression and insulin-stimulated Akt substrate of 160 kDa phosphorylation in all investigated muscles. Insulin-sensitizing effect of T0901317 was not related to changes in intramuscular level of lipid mediators of insulin resistance, since neither diacylglycerols nor ceramide content was affected by the treatment. CONCLUSION: We conclude that improvement in muscle insulin sensitivity is one of the mechanisms underlying the antidiabetic action of LXR activators.

A single prior bout of exercise protects against palmitate-induced insulin resistance despite an increase in total ceramide content.

Ceramide accumulation has been implicated in the impairment of insulin-stimulated glucose transport in skeletal muscle following saturated fatty acid (FA) exposure. Importantly, a single bout of exercise can protect against acute lipid-induced insulin resistance. The mechanism by which exercise protects against lipid-induced insulin resistance is not completely known but may occur through a redirection of FA toward triacylglycerol (TAG) and away from ceramide and diacylglycerol (DAG). Therefore, in the current study, an in vitro preparation was used to examine whether a prior bout of exercise could confer protection against palmitate-induced insulin resistance and whether the pharmacological [50 µM fumonisin B(1) (FB1)] inhibition of ceramide synthesis in the presence of palmitate could mimic the protective effect of exercise. Soleus muscle of sedentary (SED), exercised (EX), and SED in the presence of FB1 (SED+FB1) were incubated with or without 2 mM palmitate for 4 h. This 2-mM palmitate exposure impaired insulin-stimulated glucose transport (-28%, P < 0.01) and significantly increased ceramide, DAG, and TAG accumulation in the SED group (P < 0.05). A single prior bout of exercise prevented the detrimental effects of palmitate on insulin signaling and caused a partial redistribution of FA toward TAG (P < 0.05). However, the net increase in ceramide content in response to palmitate exposure in the EX group was not different compared with SED, despite the maintenance of insulin sensitivity. The incubation of soleus from SED rats with FB1 (SED+FB1) prevented the detrimental effects of palmitate and caused a redirection of FA toward TAG accumulation (P < 0.05). Therefore, this research suggests that although inhibiting ceramide accumulation can prevent the detrimental effects of palmitate, a single prior bout of exercise appears to protect against palmitate-induced insulin resistance, which may be independent of changes in ceramide content.

Restoration of skeletal muscle leptin response does not precede the exercise-induced recovery of insulin-stimulated glucose uptake in high-fat-fed rats.

Leptin administration increases fatty acid (FA) oxidation rates and decreases lipid storage in oxidative skeletal muscle, thereby improving insulin response. We have previously shown high-fat (HF) diets to rapidly induce skeletal muscle leptin resistance, prior to the disruption of normal muscle FA metabolism (increase in FA transport; accumulation of triacylglycerol, diacylglycerol, ceramide) that occurs in advance of impaired insulin signaling and glucose transport. All of this occurs within a 4-wk period. Conversely, exercise can rapidly improve insulin response, in as little as one exercise bout. Thus, if the early development of leptin resistance is a contributor to HF diet-induced insulin resistance (IR) in skeletal muscle, then it is logical to predict that the rapid restoration of insulin response by exercise training would be preceded by the recovery of leptin response. In the current study, we sought to determine 1) whether 1, 2, or 4 wk of exercise training was sufficient to restore leptin response in isolated soleus muscle of rats already consuming a HF diet (60% kcal), and 2) whether this preceded the training-induced corrections in FA metabolism and improved insulin-stimulated glucose transport. In the low-fat (LF)-fed control group, insulin increased glucose transport by 153% and leptin increased AMPK and ACC phosphorylation and the rate of palmitate oxidation (+73%). These responses to insulin and leptin were either severely blunted or absent following 4 wk of HF feeding. Exercise intervention decreased muscle ceramide content (-28%) and restored insulin-stimulated glucose transport to control levels within 1 wk; muscle leptin response (AMPK and ACC phosphorylation, FA oxidation) was also restored, but not until the 2-wk time point. In conclusion, endurance exercise training is able to restore leptin response, but this does not appear to be a necessary precursor for the restoration of insulin response.

High fat diet induces ceramide and sphingomyelin formation in rat's liver nuclei.

Obesity increases the risk for hepatic steatosis. Recent studies have demonstrated that high fat diet (HFD) may affect sphingolipid formation in skeletal muscles, heart, and other tissues. In this work we sought to investigate whether HFD feeding provokes changes in content and fatty acids (FAs) composition of sphingomyelin and ceramide at the level of liver and hepatic nuclei. Furthermore, we investigated whether the ceramide formation is related to the activity of either neutral sphingomyelinase (N-SMase) or acidic sphingomyelinase (A-SMase). Three weeks of HFD provision induced pronounced ceramide and sphingomyelin accumulation in both liver and hepatic nuclei, accompanied by increased activity of N-SMase but not A-SMase. Furthermore, a shift toward greater FAs saturation status in these sphingolipids was also observed. These findings support the conclusion that HFD has a major impact on sphingolipid metabolism not only in the liver, but also in hepatic nuclei.

Differential effects of chronic, in vivo, PPAR's stimulation on the myocardial subcellular redistribution of FAT/CD36 and FABPpm.

This study reveals that the activation of either PPARalpha (WY 14643) or PPARbeta (GW0742) each induce the translocation of FAT/CD36 from an intracellular pool(s) to the plasma membrane, while PPARbeta also induces the subcellular redistribution of FABPpm(Got2) to the plasma membrane. In contrast, activation of PPARgamma failed to induce the subcellular redistribution of FAT/CD36 and FABPpm. These PPARalpha-, and PPARbeta-induced changes in the plasmalemmal content of these fatty acid transporters were associated with the concurrent upregulation of fatty acid triacylglycerol esterification (PPARbeta) and oxidation (PPARalpha and PPARbeta). Observed effects of chronic PPAR stimulation were not related to either AMPK or ERK1/2 activation.

[The role of fatty-acid transport proteins (FAT/CD36, FABPpm, FATP) in lipid metabolism in skeletal muscles]. / Udzial bialek transportujacych (FAT/CD36, FABPpm, FATP) w metabolizmie lipidów w miesniach szkieletowych.

Skeletal muscles display an essential role in the regulation of whole-body energy homeostasis. Because of their hydrophobic nature, long-chain fatty acids (LCFAs) can enter cells via passive diffusion along the concentration gradient across the sarcolemma. However, it was also shown recently that protein-mediated transport of LCFAs occurs in skeletal muscles. So far, three groups of long-chain fatty-acid transport proteins have been identified that facilitate LCFA transport: fatty-acid translocase (FAT/CD36), plasma membrane-associated fatty-acid binding protein (FABPpm), and fatty-acid transport proteins (FATP) 1-6. Several studies revealed that both the expression and the translocation of FA transporters is process that can be highly regulated. Recent studies had shown that exercise training increases not only the oxidation of long-chain fatty acids, but also the expression of protein transporters. It was also shown that contractile activity of skeletal muscles is able to induce the translocation of protein transporters (FAT/CD36) zapotrzebofrom the intracellular compartment to the sarcolemma with a subsequent increase in LCFA transmembrane transport.