Copy numbers of OSBP/ORP mRNAs in human adipose tissues and SGBS adipocytes.

<p>The mRNAs were quantified by qPCR using the corresponding cDNAs as calibrators (see Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045352#s2" target="_blank">Methods</a>), and the mRNA quantities are presented on a log10 scale as copies/ng total RNA. A. Subcutaneous adipose tissue; B. Visceral adipose tissue. The data represents mean ± S.E., n = 4. C. Simpson-Golabi-Behmel syndrome (SGBS) adipocytes after 22-day differentiation. The mean from a single experiment carried out in triplicate is shown.</p

Time course of ORP11, ORP3, and ORP8 mRNA changes upon SGBS cell adipogenic differentiation.

<p>A. Phase contrast images of SGBS preadipocytes (0 D) and differentiating adipocytes on days 10, 14, and 22. Bars, 200 µm. B. The ORP mRNA levels were quantified at the differentiation time points indicated, as fold changes relative to preadipocytes (day 0). The data represents mean ± S.E., n = 3.</p

Impacts of ORP manipulation on SGBS cell adipogenic differentiation.

<p>ORP8 or ORP3 were overexpressed by infecting cells on day 10 with control (Blank) or ORP (Ad-ORP8, Ad-ORP3) adenoviral vectors, and collected for analysis on day 13. SGBS preadipocytes were transduced with an ORP11 shRNA (Sh-ORP11) or non-targeting (Sh-NT) shRNA lentivirus, followed by differentiation for 22 days. A. Western blots of total cell protein (10 µg/lane); ORP11, after 22 days of differentiation; ORP8 and ORP3, after 72 h of adenoviral transduction. The blots were probed with anti-β-actin as a loading control. B. The impacts of ORP11 silencing or ORP8/ORP3 overexpression on the mRNA levels of adipocyte differentiation markers adiponectin, aP2, leptin and PPARγ. In cells with ORP11 stably silenced also ORP8 and ORP3 mRNAs were quantified. The results are shown as fold changes relative to cells infected with the corresponding control viruses, and represent mean ± S.E., n = 3; *p<0.05. C. The cellular triglyceride concentration was measured by using an enzymatic assay. The results were normalized for total cell protein and are presented relative to cells infected with the corresponding control viruses (mean ± S.E., n = 3; *p<0.05, **p<0.01).</p

Adipogenic differentiation of SGBS cells.

<p>Haematoxylin and Oil Red O staining of SGBS preadipocytes (A) and adipocytes differentiated for 22 days (B). C. Relative mRNA levels of the adipocyte differentiation markers adiponectin, aP2, leptin and PPARγ in SGBS adipocytes as compared to preadipocytes. The bars indicate fold-change, mean ± S.E. (n = 3).</p

Changes in ORP mRNA and protein expression upon SGBS cell adipogenic differentiation.

<p>A. Quantities of the indicated ORP mRNAs on day 22 of differentiation. The results are presented as fold change relative to preadipocytes. The data represents mean ± S.E., n = 3−4, *p<0.05, **p<0.01. B. Western blot analysis of the ORP11, ORP8, and ORP3 proteins in SGBS preadipocytes and adipocytes (day 22). C. Quantification of the Western data by densitometric analysis. The ORP11, ORP8 and ORP3 signals were normalized for that of β-actin. The data represents mean ± S.E., n = 3−4, *p<0.05.</p

Proposed mechanism regulating LPL activity in the skeletal muscle.

<p>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.</p

Angptl4 protein expression is increased by free fatty acids and insulin in human myotubes.

<p>Concentration of (<b>a, b</b>) cell associated and (<b>c, d</b>) secreted Angptl4 protein was quantified by ELISA in myotubes derived from six men and incubated in the presence of (<b>a, c</b>) BSA-bound oleic acid (OA-BSA, 0.5 mM) or (<b>b, d</b>) Insulin (100 nM) for 24 hours. *p<0.05, paired <i>t</i> test compared with Control.</p

PPARδ activation by GW501516 inhibits LPL activity and LPL-dependent fatty acid uptake.

<p>(<b>a</b>) 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>b</b>) Time dependent inhibition of LPL activity by GW501516 (0.1 µM) was measured in heparin releasable pool from C2/LPL myotubes, n = 3. (<b>c</b>) 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. (<b>d</b>) Intracellular triglycerides quantification in cells incubated with Intralipid for 5 hours in the presence or absence of GW501516, n = 3 (<b>e</b>) 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 <i>t</i> test (<b>a</b>) or One-way ANOVA with Dunnett's post test (<b>b, c, d</b>) compared with Control.</p

Angptl4 inhibits LPL activity and LPL-dependent fatty acid uptake and mediates PPARδ effect on LPL activity.

<p>(<b>a</b>) Human Angptl4 concentration was evaluated by ELISA in medium from myotubes infected with HSA (human serum albumin)-AAV2 (Control) or hAngptl4-AAV2. (<b>b</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. (<b>c</b>) 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. (<b>d</b>) 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. (<b>e</b>) 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 <i>t</i> test (<b>b</b>) or Two-way ANOVA with Bonferroni post-tests (<b>d, e</b>) compared with Control.</p

Angptl4 overexpression has no effect on fatty acids oxidation and glucose metabolism in L6 myotubes.

<p>Fatty acid (palmitate) oxidation was evaluated by measuring (<b>a</b>) ¹⁴C-CO₂ or (<b>b</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. (<b>c</b>) 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. (<b>d</b>) Glucose uptake, (<b>e</b>) glycogen synthesis and (<b>f</b>) 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 (<b>a, b</b>) or Two-way (<b>c–f</b>) ANOVA with Bonferroni post-tests.</p