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. 2012;7(9):e45352.
doi: 10.1371/journal.pone.0045352. Epub 2012 Sep 21.

OSBP-related proteins (ORPs) in human adipose depots and cultured adipocytes: evidence for impacts on the adipocyte phenotype

You Zhou  1 , Marius R Robciuc, Martin Wabitsch, Anne Juuti, Marja Leivonen, Christian Ehnholm, Hannele Yki-Järvinen, Vesa M Olkkonen
Affiliations

OSBP-related proteins (ORPs) in human adipose depots and cultured adipocytes: evidence for impacts on the adipocyte phenotype

You Zhou et al. PLoS One. 2012.

Abstract

Oxysterol-binding protein (OSBP) homologues, ORPs, are implicated in lipid homeostatic control, vesicle transport, and cell signaling. We analyzed here the quantity of ORP mRNAs in human subcutaneous (s.c.) and visceral adipose depots, as well as in the Simpson-Golabi-Behmel syndrome (SGBS) adipocyte cell model. All of the ORP mRNAs were present in the s.c and visceral adipose tissues, and the two depots shared an almost identical ORP mRNA expression pattern. SGBS adipocytes displayed a similar pattern, suggesting that the adipose tissue ORP expression pattern mainly derives from adipocytes. During SGBS cell adipogenic differentiation, ORP2, ORP3, ORP4, ORP7, and ORP8 mRNAs were down-regulated, while ORP11 was induced. To assess the impacts of ORPs on adipocyte differentiation, ORP3 and ORP8, proteins down-regulated during adipogenesis, were overexpressed in differentiating SGBS adipocytes, while ORP11, a protein induced during adipogenesis, was silenced. ORP8 overexpression resulted in reduced expression of the aP2 mRNA, while down-regulation of adiponectin and aP2 was observed in ORP11 silenced cells. Furthermore, ORP8 overexpression or silencing of ORP11 markedly decreased cellular triglyceride storage. These data identify the patterns of ORP expression in human adipose depots and SGBS adipocytes, and provide the first evidence for a functional impact of ORPs on the adipocyte phenotype.

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Conflict of interest statement

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

Figures

Figure 1
Figure 1. Copy numbers of OSBP/ORP mRNAs in human adipose tissues and SGBS adipocytes.
The mRNAs were quantified by qPCR using the corresponding cDNAs as calibrators (see Materials and Methods), 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.
Figure 2
Figure 2. Detection of ORP proteins in human subcutaneous (S.C.) and visceral adipose tissue specimens.
Protein extracts of human adipose tissues (20µg/lane) were resolved on SDS-PAGE and analyzed by Western blotting with antibodies against OSBP, ORP2, ORP8, ORP9, ORP11, and β-actin, as indicated.
Figure 3
Figure 3. Adipogenic differentiation of SGBS cells.
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).
Figure 4
Figure 4. Changes in ORP mRNA and protein expression upon SGBS cell adipogenic differentiation.
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.
Figure 5
Figure 5. Time course of ORP11, ORP3, and ORP8 mRNA changes upon SGBS cell adipogenic differentiation.
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.
Figure 6
Figure 6. Impacts of ORP manipulation on SGBS cell adipogenic differentiation.
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).
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Grants and funding

This work was supported by the Sigrid Jusélius Foundation, the Finnish Foundation for Cardiovascular Research, the Magnus Ehrnrooth Foundation, the Liv och Hälsa Foundation, the Novo Nordisk Foundation, the Academy of Finland (grant 121457 to VMO), the EU FP7 (LipidomicNet, agreement no 202272), the Paulo Foundation (YZ), the Orion-Farmos Research Foundation (YZ), the University of Helsinki Medicine Fund and Jubilee Fund (YZ), the Finnish Atherosclerosis Society (YZ) and a University of Helsinki Chancellor travel grant (YZ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.