In the present study we have demonstrated that activation of PPARδ/RXR strongly upregulates Angptl4 causing the inhibition of LPL activity and LPL-dependent FA uptake in myotubes. In addition, we have examined the role of Angptl4 in fatty acid oxidation and glucose metabolism.
It was recently reported that in skeletal muscle cells the gene with the highest fold-induction by FAs is Angptl4 and this effect is mediated by PPARδ 
. Here we confirm a robust increase of Angptl4 by FAs or GW501516, a specific PPARδ activator 
, in both human and mouse myotubes. Processing of endogenous Angptl4 in myotubes showed a similar pattern with that observed in HEK293 or Huh7 cells overexpressing Angptl4 
, the full length, C-terminal and N-terminal forms being secreted from C2/LPL myotubes treated with GW501516. Angptl4 is a well established LPL inhibitor 
suggesting that PPARδ activation by FAs can inhibit LPL activity in myotubes. Indeed, treatment of rat and mouse myotubes with GW501516 induced a significant reduction in heparin releasable LPL activity without significant changes in LPL mRNA levels. Kinetic studies in C2/LPL cells showed a robust upregulation of Angptl4 expression already at 1 hour and a 50% reduction of LPL activity at 3 hours after the start of GW501516 treatment. Consequently, the uptake of FAs from Intralipid, which is dependent of LPL activity, was abolished by GW501516. In contrast, the uptake of free fatty acids was not affected by GW501516. Inhibition of LPL activity by PPARδ activation was dependent on Angptl4 mRNA expression since Angptl4 silencing using siRNA blocked the GW501516 effect. Previous studies have shown that in skeletal muscle LPL overexpression increases intracellular triglyceride-pool 
whereas LPL deletion decreases intracellular triglycerides stores 
. The LPL-mediated effect on lipid uptake has consequences on insulin signaling cascade in skeletal muscle 
. Since LPL activity generates FAs, it is possible that activation of the PPARδ-Angptl4 axis functions as a negative feedback mechanism that may serve to protect the muscle fibers from lipid overload (). The mechanism we suggest here is also relevant in vivo
, i.e. data provided by Tanaka et al. 
demonstrate that administration of GW501516 to mice fed a high-fat diet markedly reduced the lipid droplet formation in skeletal muscle 
. The same mechanism was recently described to protect the heart and macrophages from lipotoxicity in mice fed a high-fat diet 
In order to regulate gene expression PPARδ forms an obligatory heterodimer with RXR 
. Bexarotene is a potent and selective RXR agonist that is currently used in the treatment of cutaneous T cell lymphoma and has promising effects in other forms of cancer or dermatologic disorders but its use in clinics is limited due to its undesirable increase effect on plasma triglycerides 
. The mechanism underlying this side effect is not completely understood. Interestingly, a study performed in rats suggests that bexarotene-induced hypertriglyceridemia is attributable to inhibition of LPL activity in the muscle 
. This is further supported by the increased skeletal muscle lipoprotein lipase activity observed in RXRγ deficient mice 
. Here we found that in myotubes the inhibition of LPL activity by bexarotene is dependent on PPARδ and Angptl4. Upregulation of Angptl4 expression by bexarotene is in agreement with previous observations in thyroid cancer cell lines 
. These data suggest a mechanism that could contribute to increased triglycerides in patients during bexarotene therapy.
Population studies suggest that Angptl4 is not involved in systemic inhibition of LPL in humans 
. Since Angptl4 is the only known LPL inhibitor expressed in the same cells where LPL is produced, it is conceivable that it has more subtle roles at the tissue level. Overexpression of Angptl4 in myotubes resembles the GW501516 effect on LPL activity and LPL-dependent FA uptake. Angptl4 acts in a concentration dependent manner and interestingly, it appears more effective when expressed together with LPL within the same cells than when added as a recombinant protein. A weaker effect of Angptl4 on LPL activity was also seen in cells treated with conditioned medium from myotubes overexpressing Angptl4 (data not shown). This phenomenon can be explained by our observation that Angptl4 inhibited LPL activity not only at the cell surface, represented by the heparin releasable pool, but also intracellularly. Moreover, confocal microscopy analysis revealed extensive intracellular co-localization of LPL and Angptl4 in reticular perinuclear-concentrated ER membranes. All these observations suggest that LPL and Angptl4 could interact intracellularly making the inhibition more efficient when both proteins are expressed within the same cell. The inhibition of LPL by Angptl4 occurs before GPIHBP1 interaction with LPL and its transport to the luminal site of the capillary endothelium 
. This notion is physiologically relevant since GPIHBP1 was shown to prevent LPL inhibition by Angptl4 
. Interestingly, insulin increases Angptl4 expression in myotubes which is in contrast with adipocytes where insulin downregulates Angptl4 mRNA expression 
. All these data suggest that Angptl4 could be implicated in the tissue specific regulation of LPL activity by insulin 
PPARδ overexpression or activation by GW501516 increased the catabolism of FAs by upregulation of genes implicated in FA uptake, handling, and mitochondrial import in skeletal muscle 
. Because Angptl4 is a multifunctional protein we tested whether it plays a role in fatty acid (palmitate) oxidation. As expected, PPARδ activation increased 14
C-ASM production by L6 myotubes, however, Angptl4 overexpression had no effect on the β-oxidation. We confirmed this by using 3
H-palmitic acid and quantification of the 3
O produced by L6 myotubes overexpressing Angptl4. Angptl4 was suggested to upregulate lipolytic enzymes such as hormone sensitive lipase in C2C12 myocytes 
and therefore we tested wheather Angptl4 could enhance the oxidative capacity of GW501516. Using a low dose of GW501516 we observed a similar induction of palmitate oxidation in control and Angptl4 overexpressing L6 myotubes. These data strongly suggest that the induction of FA oxidation by PPARδ is not dependent on extra- or intracellular Angptl4.
Previous studies have shown that Angptl4 significantly decreased hepatic glucose production and enhanced insulin-mediated inhibition of gluconeogenesis suggesting a role of Angptl4 in glucose metabolism 
. In our study, Angptl4 overexpression in L6 myotubes neither affected glucose uptake, glycogen synthesis and glucose oxidation, nor insulin function in regulating these processes. These results suggest a tissue specific regulation of glucose metabolism by Angptl4.
A limitation to consider in our study is that we used cultured myotubes and this system lacks the endothelial cell layer where LPL exerts its function in vivo. Because Angptl4 and LPL are produced in the same cells it is relevant that their interaction should occur before LPL reaches endothelial cells. For this purpose myotubes provide a relevant model to study the mechanism of LPL inhibition by Angptl4 in contrast to liver derived inhibitors that probably act mainly at the luminal surface of the capillary endothelium.
In conclusion, our principal results suggest that an overflow of FAs inhibits LPL activity in skeletal muscle. The working hypothesis is that FAs produced locally via the LPL function or released from adipose tissue as albumin bound FAs can activate PPARδ/RXR heterodimer which in turn upregulates Angptl4 gene expression. Angptl4 inhibits LPL activity mainly at the surface of the sarcolemma where less LPL will be available to be transported at luminal sites via the function of GPIHBP1 and therefore the flow of FAs in the tissue is reduced (). This mechanism may also contribute to increased plasma triglycerides frequently observed in patients treated with bexarotene. In addition, PPARδ increases the FA oxidation capacity of the tissue independently of Angptl4. All these suggest PPARδ as being a key player in maintaining the balance between the FA uptake and oxidation capacity of the skeletal muscle.
Proposed mechanism regulating LPL activity in the skeletal muscle.