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

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

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

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

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

PPARδ activation increases Angptl4 mRNA and protein levels in human and mouse C2/LPL myotubes.

<p>(<b>a</b>) 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>b</b>) human and (<b>c</b>) 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. (<b>d</b>) 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 <i>t</i> test compared with Control.</p

Regulation of Angptl4 expression and LPL activity by bexarotene in C2/LPL myotubes.

<p>(<b>a</b>) 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>b</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. (<b>c</b>) 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.</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