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Both LXR isoforms were shown to be expressed in human and rodent skeletal muscle 12, To date only a few studies performed on cultured myotubes have dealt with the effect of T on muscle metabolism. However, considering the fact that in vivo LXR activation can dramatically alter plasma lipid profile, there is a need for re-evaluation of the action of LXR agonists on muscle tissue in whole-animal model. In the present study we characterize for the first time in details the effect of in vivo activation of LXR on lipid metabolism in skeletal muscle.
The dosage and the length of treatment were chosen after Cao et al. They also found that one week treatment with the drug was sufficiently long to induce strong effects on lipid metabolism in rats. The solutions were administrated once daily in the morning by an oral gavage. On the last day of the experiment, between 8 and 10 a. Samples of the soleus as well as red and white sections of the gastrocnemius muscle were excised and immediately freeze-clamped with aluminum tongs precooled in liquid nitrogen.
Blood taken from the abdominal aorta was collected in heparinized tubes, centrifuged, the plasma separated and flash-frozen in liquid nitrogen. Muscle lipids Samples were pulverized in an aluminum mortar precooled in liquid nitrogen. Lipids were extracted by the method of Folch.
The fractions of total phospholipids, triacylglycerols, diacylglycerols, nonesterified fatty acids NEFA , free cholesterol and cholesterol esters were separated by thin-layer chromatography TLC according to Roemen and van der Vusse The content of resulting fatty acid methyl esters was determined using gas-liquid chromatography as previously described in detail Free cholesterol and cholesterol esters were eluted from the gel with chloroform, evaporated under nitrogen stream and redissolved in 2-propanol or diethyl ether, respectively.
The content of free cholesterol and cholesterol esters was subsequently measured with commercially available cholesterol diagnostic kit BioMaxima. The content of ceramide was determined as described previously in detail Briefly, tissue lipids were extracted into chloroform and the samples were then subjected to alkaline hydrolysis to deacylate ceramide.
Free sphingosine liberated from ceramide was converted to o-phthalaldehyde derivative and analyzed using HPLC system. N-palmitoyl-D-erythro-sphingosine C17 base a kind gift of Dr. Szulc, Medical University of South Carolina was used as an internal standard.
Soleus muscle incubations Palmitate oxidation and esterification in incubated soleus muscle strips was determined using [9,H]-palmitate, as previously described in detail Palmiate oxidation was estimated by measuring the release of 3H2O into the incubation buffer.
To determine palmitate esterification, lipids were extracted from muscle strips and then separated by means of TLC. The lipid bands were scraped off the plates, and 3H-palmitate incorporation into different lipid pools was measured by radioactivity counting.
The quality of each RNA sample was verified by running the agarose electrophoresis with ethidium bromide. Oligonucleotide primers were designed using Beacon Designer Software 7. PCR efficiency was examined by serially diluting the template cDNA, and a melt curve was performed at the end of each reaction to verify PCR product specificity. A sample containing no cDNA was used as a negative control to verify the absence of primer dimers.
The results were normalized to b-actin expression measured in each sample. After incubation with the secondary alkaline phosphatase-conjugated antibody Sigma, A protein bands were scanned and quantified using a Gel Doc EQ system Bio-Rad.
Plasma NEFA and triacylglycerols concentration was markedly increased by T, whereas total cholesterol level was reduced by the drug. Moreover, LXR activation resulted in a slight decrease in plasma glucose concentration Table 1.
Table 1. General features and plasma measurements of the experimental animals. NEFA - nonesterified fatty acids. Animals treated with T were characterized by decreased muscle content of NEFA and free cholesterol.
There was also a marked reduction in the level of cholesterol esters, however, this effect was observed only in the soleus and red gastrocnemius Fig. On the other hand, the content of phospholipids was elevated upon LXR activation in all examined muscles. In addition, administration of T increased ceramide level in the soleus Fig.
Animals treated with the LXR agonist were also characterized by accumulation of triacylglycerols in the red gastrocnemius but not in the soleus or white gastrocnemius, where no statistically significant changes were observed.
The content of diacylglycerols in the soleus and red gastrocnemius was not affected by T, whereas, in the white gastrocnemius it was reduced upon LXR activation Fig. Effect of LXR activation on muscle content of major lipid classes. It should be noted, however, that in the animals administered with T this ratio was markedly higher in the red gastrocnemius compared to the soleus and white gastrocnemius Fig.
Expression of LXR in rat skeletal muscles. Table 2. Effect of T on mRNA level of selected genes in skeletal muscle. Upregulation of SREBP-1 protein was more pronounced in the soleus as compared to the other two examined muscles.
Interestingly, expression of diacylglycerol acyltransferase DGAT 1 was strongly suppressed in all examined muscles by T Table 2. It should be noted, however, that expression of MCD was upregulated only in the soleus and red gastrocnemius Table 2. Effect of T on protein expression of selected genes in skeletal muscle. Strips of the soleus muscle isolated from Ttreated animals were characterized by lower rate of 3H-palmitate incorporation into triacylglycerols, whereas the rate of its incorporation into phospholipids was increased.
There were no statistically significant changes in the amount of tracer incorporated into intramuscular NEFA or diacylglycerols Fig. The rate of 3H-palmitate oxidation in the soleus muscle strips isolated from rats administered with LXR agonist was markedly higher as compared to the vehicle control Fig.
LXR activation enhanced palmitate oxidation in isolated soleus muscle strips. Strips of the soleus muscle were then isolated and incubated with [3H]-palmitate as described in Materials and Methods. A incorporation of palmitate into intramuscular lipids, B palmitate oxidation to H2O. However, palmitate incorporation into triacylglycerol was decreased, which was associated with reduced DGAT expression.
Despite markedly increased availability of plasma lipids upon T treatment, muscle triacylglycerol was elevated only in red gastrocnemius. Our results indicate significant differences in LXR expression between muscles with different fiber type composition. Namely, protein level of both LXR isoforms was higher in muscles with high oxidative capacity soleus and red gastrocnemius compared to the glycolytic one white gastrocnemius.
A similar pattern was reported for PPARs expression in rat skeletal muscles There is, however, conflicting evidence in the literature as to whether LXR activation enhances expression of other lipogenic genes in muscle cells.
In our study, SCD1 expression was strongly induced in all muscles upon T administration. The same finding was reported for cultured human myotubes treated with T This is in contrast to a response observed in the liver, where all lipogenic genes are coordinately and strongly upregulated upon LXR activation 9, Liang et al.
Therefore, our results suggest that limited response of lipogenic pathway to synthetic LXR agonists, that is observed in skeletal muscle, is a consequence of their inability to effectively upregulate mature SREBP-1c protein therein.
We observed a significant increase in plasma triacylglycerols concentration after treatment with T Hypertriglyceridemia is a well-documented effect of T in rodents, and results from augmented hepatic very low density lipoprotein VLDL -triglyceride secretion It should be noted that hypertriglyceridemic effect of T in mice is transient and limited to the first three days of the treatment 27, In our study, it was still present after one week which provides additional evidence for the presence of differences between rats and mice in response to synthetic LXR agonists.
Similar finding was reported by other groups 28, It was shown that LXR activation stimulates basal lipolysis in human adipocytes, and we have previously observed increased expression of hormone-sensitive lipase and adipocyte triglyceride lipase in adipose tissue of rats treated with T On the other hand, there was no clear tendency towards increased content of muscle lipids upon T administration.
We observed a decrease in intramuscular NEFA level, and no change soleus and red gastrocnemius or a decrease white gastrocnemius in diacylglycerols concentration.
Triacylglycerols were increased exclusively in red gastrocnemius, and only phospholipids content was moderately elevated in all examined muscles. There is very few data on the effect of LXR agonists on lipid content in muscle tissue. Hessvik et al. However, Cozzone et al. Korach-Andre et al. This observation is in good agreement with our results, as we observed no effect of T on triacylglycerols level in white gastrocnemius, a muscle with fiber composition similar to murine tibialis anterior Similarly, Commerford et al.
Unfortunately, the authors did not indicate which portion of the gastrocnemius muscle they had used. A surprising finding of our study is that T did not cause overt accumulation of intramuscular lipids despite significant increase in plasma NEFA and triacylglycerols concentration and enhanced muscle expression of genes involved in lipogenesis and fatty acid uptake.
This observation suggests that, in contrast to liver which becomes fatty and enlarged, muscle lipid homeostasis is not perturbed by pharmacological LXR activation. Our results indicate that T not only increased fatty acid availability in skeletal muscle but also stimulated their oxidation therein, which prevented accumulation of lipids. In addition, plamitate oxidation rate was markedly elevated in the soleus muscle strips isolated from rats administered with the LXR agonist.
This finding is consistent with our previous report showing that T increased muscle fatty acid utilization during exercise The fatty acyl composition of PLs determines the biophysical characteristics of membranes, including fluidity and the assembly of specific membrane subdomains Holzer et al. Therefore, changes in fatty acyl composition can affect the properties of proteins associated with membranes and influence the biological processes that occur on them.
Modification of the fatty acyl composition of membranes influences a range of cell processes, most importantly, the activity of membrane-bound enzymes and transporters and the localization of acylated proteins in membrane subdomains Cornelius, ; Fu et al.
It is also known that incorporation of saturated fatty acids into plasma membrane recruits c-Src kinase to lipid raft domains and increases its activity Holzer et al. In mammalian cells, PLs are initially synthesized by the de novo pathway and subsequently undergo remodeling through fatty acyl deacylation and reacylation, a pathway referred to as the Lands cycle Lands, As a result, saturated fatty acids are preferably linked at the sn-1 position and unsaturated fatty acids at the sn-2 position.
This diversity and asymmetric distribution is established largely by the remodeling process, as the de novo PL synthesis process has little fatty acyl-CoA substrate specificity. In the liver, a major enzyme that catalyzes the formation of phosphatidylcholine PC from saturated lysophosphatidylcholines LysoPC and unsaturated fatty acyl-CoAs is lysophosphatidyl acyltransferase 3 Lpcat3 Hishikawa et al. Lpcat3 preferentially synthesizes PC containing unsaturated fatty acids, particularly arachidonic acid To date, most studies of the effects of PL fatty acyl composition on biological systems have utilized in vitro biochemical assays, due to the difficulty of directing specific changes in membrane composition in living cells.
Therefore, there is little understanding of how regulatory pathways control PL fatty acyl composition or how such regulatory pathways could dictate cell responses. It is known that increased levels of saturated fatty acids can cause ER stress, and this has been postulated to involve charges in ER membrane composition Borradaile et al. It is also been shown that inhibition of SCD-1 or Lpcat3 activity increases membrane saturation, secondary to changes in fatty acid production Miyazaki et al.
But are there regulatory pathways that modify membrane lipid composition in response to extracellular or intracellular cues? Furthermore, could such pathways be targeted pharmacologically to manipulate ER membrane composition? Finally, what is the contribution of PL remodeling to ER stress responses in the setting of metabolic disease? The Liver X receptors LXRs are important regulators of cholesterol and fatty acid homeostasis and potent inhibitors of inflammation Hong and Tontonoz, However, the impact of LXRs on the major constituents of membranes—phospholipids—has not been rigorously investigated.