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. 2012;7(10):e46212.
doi: 10.1371/journal.pone.0046212. Epub 2012 Oct 4.

Angiopoietin-like 4 mediates PPAR delta effect on lipoprotein lipase-dependent fatty acid uptake but not on beta-oxidation in myotubes

Marius R Robciuc  1 , Paulina Skrobuk, Andrey Anisimov, Vesa M Olkkonen, Kari Alitalo, Robert H Eckel, Heikki A Koistinen, Matti Jauhiainen, Christian Ehnholm
Affiliations

Angiopoietin-like 4 mediates PPAR delta effect on lipoprotein lipase-dependent fatty acid uptake but not on beta-oxidation in myotubes

Marius R Robciuc et al. PLoS One. 2012.

Abstract

Peroxisome proliferator-activated receptor (PPAR) delta is an important regulator of fatty acid (FA) metabolism. Angiopoietin-like 4 (Angptl4), a multifunctional protein, is one of the major targets of PPAR delta in skeletal muscle cells. Here we investigated the regulation of Angptl4 and its role in mediating PPAR delta functions using human, rat and mouse myotubes. Expression of Angptl4 was upregulated during myotubes differentiation and by oleic acid, insulin and PPAR delta agonist GW501516. Treatment with GW501516 or Angptl4 overexpression inhibited both lipoprotein lipase (LPL) activity and LPL-dependent uptake of FAs whereas uptake of BSA-bound FAs was not affected by either treatment. Activation of retinoic X receptor (RXR), PPAR delta functional partner, using bexarotene upregulated Angptl4 expression and inhibited LPL activity in a PPAR delta dependent fashion. Silencing of Angptl4 blocked the effect of GW501516 and bexarotene on LPL activity. Treatment with GW501516 but not Angptl4 overexpression significantly increased palmitate oxidation. Furthermore, Angptl4 overexpression did not affect the capacity of GW501516 to increase palmitate oxidation. Basal and insulin stimulated glucose uptake, glycogen synthesis and glucose oxidation were not significantly modulated by Angptl4 overexpression. Our findings suggest that FAs-PPARdelta/RXR-Angptl4 axis controls the LPL-dependent uptake of FAs in myotubes, whereas the effect of PPAR delta activation on beta-oxidation is independent of Angptl4.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Angptl4 mRNA and protein levels are increased during differentiation of myotubes.
Proliferative myoblasts from human (a) and mouse (d) C2/LPL were differentiated in multinucleated (b, inset) myotubes (b, human and e, mouse) and visualized using conventional light microscopy. (c) Secretion of Angptl4 was quantified by ELISA from day 2 to day 7 of differentiation and adjusted for protein content in cells derived from three men. (f) Angptl4 and PPARδ mRNA levels were measured by real time PCR during differentiation of C2/LPL myoblasts, n = 4. Values are expressed as fold increase relative to day two of differentiation and normalized to human β-actin or mouse 36B4 mRNA levels. Results are expressed as mean ± SEM, (c) n = 3 and (f) n = 4. Scale bar: 50 µm. *p<0.05 as compared with day 2 using Repeated Measures ANOVA with Dunnett's post test.
Figure 2
Figure 2. Angptl4 protein expression is increased by free fatty acids and insulin in human myotubes.
Concentration of (a, b) cell associated and (c, d) secreted Angptl4 protein was quantified by ELISA in myotubes derived from six men and incubated in the presence of (a, c) BSA-bound oleic acid (OA-BSA, 0.5 mM) or (b, d) Insulin (100 nM) for 24 hours. *p<0.05, paired t test compared with Control.
Figure 3
Figure 3. PPARδ activation increases Angptl4 mRNA and protein levels in human and mouse C2/LPL myotubes.
(a) Human myotubes were incubated for 24 hours in the presence of DMSO (Control) or GW501516 (0.1 µM) and Angptl4 concentration in medium and cell lysate was quantified using ELISA, n = 4. Angptl4 mRNA levels were measured by real time PCR in (b) human and (c) mouse myotubes incubated with DMSO (Control) or GW501516 (0.1 µM) for 24 hours, n = 4. Angptl4 mRNA levels were normalized and analyzed in parallel with human PBGD and mouse HRPT mRNA levels. (d) Secretion of Angptl4 from C2/LPL myotubes was analyzed by Western blot 48 hours after incubation with DMSO (Control) or GW501516 (0.1 µM). Samples from two experiments were loaded on the gel. Right panel shows Ponceau staining of the blotting membrane as a control for protein loading and efficient transfer from the gradient gel to nitrocellulose membrane. *p<0.05, paired t test compared with Control.
Figure 4
Figure 4. PPARδ activation by GW501516 inhibits LPL activity and LPL-dependent fatty acid uptake.
(a) Heparin releasable LPL activity was measured in the L6, C2C12 and C2/LPL myotubes after 24 hour incubation of the cells in the presence of GW501516, n = 3. (b) Time dependent inhibition of LPL activity by GW501516 (0.1 µM) was measured in heparin releasable pool from C2/LPL myotubes, n = 3. (c) Oil Red O staining of myotubes was quantified by densitometry in cells incubated with Intralipid or OA-BSA for 5 hours in the presence or absence of GW501516, n = 4. (d) Intracellular triglycerides quantification in cells incubated with Intralipid for 5 hours in the presence or absence of GW501516, n = 3 (e) Fluorescence microscope images of C2/LPL myotubes (highlighted with a line) incubated with Intralipid for 5 hours in the presence or absence of GW501516. Nuclei are stained in blue (DAPI) and lipid droplets in red (Oil Red O). * p<0.05, paired t test (a) or One-way ANOVA with Dunnett's post test (b, c, d) compared with Control.
Figure 5
Figure 5. Regulation of Angptl4 expression and LPL activity by bexarotene in C2/LPL myotubes.
(a) Secretion of Angptl4 from C2/LPL myotubes was analyzed by Western blot 48 hours after incubation with DMSO (Control) or 0.2 µM bexarotene. Samples from two experiments were loaded on the gel. (b) Angptl4 and LPL mRNA levels were measured by real time PCR in C2/LPL myotubes incubated with DMSO (Control) or 0.2 µM bexarotene for 24 hours. Values are expressed relative to mouse 36B4 mRNA levels. (c) C2/LPL myotubes untreated or pre-treated overnight with 1 µM GSK0660, a PPARδ antagonist, were incubated with DMSO (Control) or 0.2 µM bexarotene for 4 hours. Heparin releasable LPL activity was measured and normalized to the protein content. * p<0.05, Two-way ANOVA with Bonferroni post-tests.
Figure 6
Figure 6. Angptl4 inhibits LPL activity and LPL-dependent fatty acid uptake and mediates PPARδ effect on LPL activity.
(a) Human Angptl4 concentration was evaluated by ELISA in medium from myotubes infected with HSA (human serum albumin)-AAV2 (Control) or hAngptl4-AAV2. (b) Heparin releasable LPL activity was measured in myotubes infected with HSA-AAV2 (Control) or hAngptl4-AAV2, n = 4 for L6 and n = 3 for C2/LPL. LPL activity expressed as 100% represent 0.037 (L6) or 3.38 (C2/LPL) µmol FAs h−1 mg−1. (c) Heparin releasable LPL activity was measured in C2/LPL myotubes exposed to increasing concentration of recombinant hAngptl4. LPL activity expressed as 100% represent 2.58 µmol FAs h−1 mg−1. (d) Myotubes infected with HSA-AAV2 or Angptl4-AAV2 or treated with Orlistat (50 µM) were incubated 16 h with Intralipid or OA-BSA. Oil Red O staining of myotubes was quantified by densitometry, n = 4. (e) Cells transfected with non targeting siRNA (NT-siRNA) or Angptl4 siRNA were incubated with GW501516 for 4 hours and heparin releasable LPL activity was quantified, n = 3. * p<0.05, paired t test (b) or Two-way ANOVA with Bonferroni post-tests (d, e) compared with Control.
Figure 7
Figure 7. Angptl4 inhibits intracellular LPL activity and co-localizes with intracellular LPL.
(a) Heparin releasable and intracellular LPL activity was measured in C2/LPL myotubes incubated with GW501516 (0.1 µM) for 24 hours, n = 3. (b, c, d) Confocal microscopy of myoblasts transfected with V5 tagged Angptl4 and stained with antibodies against LPL (b) and V5 tag (c). Scale bar: 20 µm. * p<0.05, paired t test.
Figure 8
Figure 8. Angptl4 overexpression has no effect on fatty acids oxidation and glucose metabolism in L6 myotubes.
Fatty acid (palmitate) oxidation was evaluated by measuring (a) 14C-CO2 or (b) 14C-acid soluble metabolites (14C-ASM) production in L6 myotubes 72 hours post-infection with HSA-AAV2 (Control) or Angptl4-AAV2 and compared with cells incubated with DMSO (Control) or GW501516 for 24 hours, n = 3. (c) Palmitate oxidation was evaluated by measuring 3H-H2O released from myotubes incubated with low doses of GW501516 in the setting of low (HSA-AAV2) and high (Angptl4-AAV2) Angptl4 levels, n = 4. (d) Glucose uptake, (e) glycogen synthesis and (f) glucose oxidation were measured in L6 myotubes 72 hours post-infection with HSA-AAV2 or Angptl4-AAV2 in the absence (Basal) or presence of insulin (100 nM), n = 3. *p<0.05, One-way (a, b) or Two-way (c–f) ANOVA with Bonferroni post-tests.
Figure 9
Figure 9. Proposed mechanism regulating LPL activity in the skeletal muscle.
The working hypothesis is that FAs produced locally by LPL-mediated hydrolysis of VLDL and chylomicrons (CM) together with FAs derived from adipose tissue (FA-albumin) 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. LPL inhibition by Angptl4 occurs to a lesser extent also intracellularly. This mechanism may protect the skeletal muscle from lipid overload and insulin resistance but may also contribute to bexarotene induced systemic hypertriglyceridemia.
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