The authors have declared that no competing interests exist.
Conceived and designed the experiments: MW CE HY-J VMO. Performed the experiments: YZ MRR. Analyzed the data: YZ VMO. Contributed reagents/materials/analysis tools: AJ ML. Wrote the paper: YZ MRR HY-J VMO.
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.
Bioactive lipid signaling molecules are key players in cell regulation, and disturbances in signaling lipids such as oxysterols, sphingolipids/ceramides, fatty acid derivatives and diacylglycerols, are associated with metabolic and cardiovascular diseases
Oxysterols are 27-carbon oxygenated products of cholesterol that arise through enzymatic or non-enzymatic oxidation processes, or are absorbed from the diet
Oxysterol-binding protein (OSBP) and its homologues designated OSBP-related proteins (ORPs) are lipid binding proteins, the β-barrel-like ligand-binding domain of which can accommodate lipids such as oxysterols, cholesterol, or phosphatidylinositol-4-phosphate
The rabbit ORP2, ORP3, ORP8 and ORP11 antibodies have been described previously
mRNA | Forward primer 5′-3′ | Reverse primer 5′-3′ |
OSBP |
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ORP1S |
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ORP1L |
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ORP2 |
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ORP3 |
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ORP4 |
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ORP5 |
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ORP6 |
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ORP7 |
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ORP8 |
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ORP9 |
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ORP10 |
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ORP11 |
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Adiponectin |
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aP2 |
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Leptin |
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PPARγ |
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SDHA |
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β-actin |
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Protein samples for SDS-PAGE were prepared by homogenizing cultured cells in 250 mM Tris-HCl, pH 6.8, 8% SDS, protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). The crude extracts were cleared by centrifugation in a microfuge at 13,000 rpm for 3 min, and the protein concentration of the supernatants was determined by the DC assay (BioRad, Hercules, CA). The proteins were electrophoresed on Laemmli gels and electrotransferred onto Hybond-C Extra nitrocellulose membranes (Amersham Biosciences, Piscataway, NJ) for different periods of time according to the protein size. Unspecific binding of antibodies was blocked with, and all antibody incubations were carried out in 5% fat free powdered milk in 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.05% Tween 20. The bound primary antibodies were visualized with horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad) and the enhanced chemiluminescence system (Thermo Scientific, Rockford, IL). The expression levels were quantified by densitometry using ImageJ (
The mRNAs were quantified by qPCR using the corresponding cDNAs as calibrators (see Materials and
Protein extracts of human adipose tissues (20
Human Simpson-Golabi-Behmel syndrome (SGBS) pre-adipocytes
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).
pGIPZ-ORP11 shRNA construct (Cat. no. RHS4430-98704927) and pGIPZ empty vector were purchased from OpenBioSystems/Thermo Scientific (Huntsville, AL). Lentiviral particles were generated by transfection of the shRNA and packing plasmids pHelper and pEnvelope to HEK 293FT cells using the FuGENE HD Reagent (Roche Diagnostics). After three days’ transfection, the viral supernantant was filtered through a 0.45 µm PVDF filter and further concentrated by ultracentrifugation for 2 hrs in an SW28 rotor at 19,400 rpm, 4°C. ORP3 and ORP8 adenoviruses were produced as described
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.
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.
The microarray dataset of Human U133A/GNF1H Gene Atlas from BioGPS web site (
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).
Human subcutaneous and visceral adipose tissue specimens were obtained from four morbidly obese patients upon bariatric surgery at Peijas Hospital, Finland. The patients were female and their ages ranged from 33 to 64 yrs. Their average BMI was averaged 46.9 kg/m2 (range 43.7 to 50.8 kg/m2) and waist to hip ratio 0.91 (range 0.88–0.96).
The subjects were volunteers recruited according to a permission (28/13/03/02/2010) from the Medicinal Ethics Committee of the Helsinki and Uusimaa Hospital Distric. The Finnish Ethics Committees obey the current Finnish law. The Medical Research Act No. 488/1999 takes into consideration statutory regulations and directives (Mostly Directives 2001/20/EC and 2005/28/EC) within EC area. The Ethics committee operates according to the principles of good clinical research practice (ICH-GCP-E6) and in accordance with the international obligations concerning the status of re- search subjects and the rules and guidelines that govern research (Medical Research Act 488/1999, chapter 2 a (23.4.2004/295), section 5 and 10a). The Medicinal Ethics Committee of the Helsinki and Uusimaa Hospital District is registered with the HHS and follows ICH-GCP-E6 in reviewing all research in the Helsinki and Uusimaa Hospital District involving human subjects. Minerva Foundation Institute for Medical Research does not have its own ethics committee. All clinical research carried out by Minerva researchers occurs at the clinics of the Helsinki and Uusimaa Hospital District, and ethical approvals for these studies have to be exclusively obtained from the Medicinal Ethics Committee of this Hospital District. No research was conducted outside our country of residence. The patients signed a written consent after receiving detailed information on the study protocol and after having an adequate time for deliberation. All procedures and possible hazards, risks and discomforts were explained to the subjects both orally and in writing. The signed forms are archived in our study documentation.
Total RNA (RNeasy Lipid Tissue Mini kit, QIAGEN, Germantown, MD) was extracted from s.c. and visceral adipose tissues of 4 patients. The RNAs were reverse transcribed by using the VILO kit (Invitrogen, Carlsbad, CA) and subjected, by using primer sets specific for 13 major ORP transcripts (
Human ORP open reading frames were cloned into pcDNA4HisMaxC (Invitrogen, Carlsbad, CA) as described before
Cells were washed with PBS, fixed by using 10% formalin for 30 min and then gently rinsed with water. After incubation with 60% isopropanol for 5 min, the cellular neutral lipids were stained by using 1.8 mg/ml Oil Red O in 60% isopropanol. Cells were rinsed with water and stained by using Hematoxylin at room temperature for 1 min.
After lentiviral or adenoviral transduction, SGBS adipocytes were lysed in RIPA buffer containing 50 mM Tris-HCl (pH 7,4), 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1% SDS and protease inhibitor cocktails (Roche Diagnostics). The supernatant was harvested after centrifugation for 5 min, and 10 µl samples as well as the glycerol standards (2300, 1150, 575, 287.5, 143.75 and 71.88 µmol/L) were subsequently mixed with 200 µl of the GPO-PAP Triglyceride kit (Cobas, Roche/Hitachi) reagent on a 96-well plate. After incubation at room temperature for 20 min, colorimetric assay was performed at 510 nm by using a Victor multilabel analyzer (Wallac, Turku, Finland). The triglyceride concentrations were normalized for total cell protein determined with the DC assay (BioRad).
The data are presented as mean ± standard error of mean (SEM). Student’s T-test was applied for comparisons between groups of data points. All statistical tests were two-sided and p values <0.05 were considered significant.
OSBP/ORP mRNA copy numbers were determined in s.c. and visceral adipose tissue biopsy specimens from 4 obese female patients as well as in SGBS adipocytes. The OSBP/ORP expression patterns of the s.c. and visceral fat depots were almost identical (
By applying Human U133A/GNF1H Gene Atlas dataset, we investigated OSBP/ORPs expression profiles across the human tissues with the help of a heat-map (
To assess the OSBP/ORP expression level during adipocyte differentiation, we employed the SGBS preadipocyte-adipocyte model. After 22 days of differentiation, approximately 80% of the cells were filled by large lipid droplets (
We analyzed ORP mRNA relative quantities in the preadipocytes and adipocytes on day 22. Quantitative RT-PCR analyses revealed that the ORP2 (−54%), ORP3 (−86%), ORP4 (−74%), ORP7 (−56%), and ORP8 (−67%) messages were significantly down-regulated during SGBS cells adipogenesis, while the ORP11 mRNA was elevated 3.5-fold (
To determine the time course of the alterations in the mRNA levels of ORP3, ORP8, and ORP11 during SGBS cell adipogenesis, we analyzed the mRNA expression levels on days 0, 4, 10, 14, 16, 18, and 22 of differentiation. The number and size of lipid droplets in the SGBS cells increased gradually (
To elucidate whether ORP3, ORP8, and ORP11 influence adipogenesis in SGBS cells, we silenced ORP11, the protein up-regulated upon SGBS adipocyte differentiation, by using a lentiviral shRNA expression vector. Correspondingly, we overexpressed two ORPs down-regulated upon adipocyte differentiation, ORP3 and ORP8, by using recombinant adenoviral vectors.
We first established SGBS preadipocytes transduced with a lentivirus expressing ORP11 shRNA; 60–70% reduction of the ORP11 protein was observed in SGBS adipocytes differentiated from the transduced preadipocytes, as compared to the controls transduced with a non-targeting shRNA lentivirus (
ORP3 or ORP8 were overexpressed for 3 days starting on day 10 of SGBS adipocytic differentiation, a time at which the endogenous ORP3 and −8 mRNAs were firmly down-regulated and the cytoplasmic lipid droplets were rapidly growing (see
To monitor the putative impacts of the ORP manipulations on the hallmark of the adipocyte phenotype, neutral lipid accumulation, we quantified triglyceride concentrations in SGBS cells in which ORP11 was silenced, on day 22 of differentiation, and in cells transduced for 3 days with ORP3 or ORP8 adenoviruses as specified above. This analysis revealed that silencing of ORP11 and overexpression of ORP8 markedly (by >50%) reduced the storage of cellular triglycerides as compared to cells transduced with the appropriate control viruses, while ORP3 overexpression did not significantly influence the cellular triglyceride accumulation (
Members of the ORPs family are thought to act as sterol sensors that relay information to the cellular machineries maintaining lipid homeostasis, energy balance and cell signaling. In the present study, we describe OSBP/ORP expression patterns in human adipose depots and SGBS preadipocytes/adipocytes. Furthermore, we present evidence for a functional impact of ORPs on the adipocyte phenotype.
OSBP/ORPs displayed a similar expression pattern in subcutaneous and visceral adipose tissues from morbidly obese patients. However, the expression profiles across different ORPs were markedly uneven, and resembled that in SGBS adipocytes. This suggests that the distinct ORP expression pattern observed in the human adipose tissues mainly reflects the adipocytic ORP profile, and is not strongly disturbed by other cell types present in the fat tissues, such as fibroblasts, macrophages, and endothelial cells
ORP11 was reported to be up-regulated in the visceral adipose tissue of obese Canadian men with metabolic syndrome compared to men without the metabolic syndrome
ORP8 is abundantly expressed in CD14(+) monocytes (
ORP3 overexpression did not significantly alter the adipocyte differentiation marker mRNA levels or the triglyceride storage in SGBS adipocytes. This speaks against an important role of ORP3 in the control of adipocyte differentiation. We previously characterized ORP3 as a phosphoprotein involved in cell adhesion, capable of modifying integrin activity, the actin cytoskeleton, and phagocytosis
ORP2 was recently shown to have the capacity to facilitate cholesterol transfer from the plasma membrane to the endoplasmic reticulum and lipid droplets
In summary, we report the expression patterns of OSBP/ORPs in human s.c. and visceral adipose depots and present, by using the SGBS preadipocyte/adipocyte model, the first data on the functional role of ORP proteins in adipocyte differentiation.
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We are grateful to Eeva Jääskeläinen, Liisa Arala, Anne Salo and Mia Urjansson for skilled technical assistance. Prof. Maria Antonietta De Matteis is thanked for kindly providing the human OSBP cDNA and antibody, Prof. Neale Ridgway for the ORP9 antibody, Dr. Daniel Tews for valuable advice on the culture of SGBS cells, and Dr. Janne Makkonen for his contribution to the patient recruitment.