Yonsei Med J.  2007 Oct;48(5):765-772. 10.3349/ymj.2007.48.5.765.

Expression and Regulation of Osteoprotegerin in Adipose Tissue

• An JJ
• Han DH
• Kim DM
• Kim SH
• Rhee Y
• Lee EJ
• Lim SK

• KMID: 1122612
• DOI: http://doi.org/10.3349/ymj.2007.48.5.765

Abstract

PURPOSE: Osteoprotegerin (OPG), a potent inhibitor of osteoclastic bone resorption, has a variety of biological functions that include anti-inflammatory effects. Adipocytes and osteoblasts share a common origin, and the formation of new blood vessels often precedes adipogenesis in developing adipose tissue microvasculature. We examined whether OPG is secreted from adipocytes, therefore contributing to the prevention of neovascularization and protecting the vessels from intimal inflammation and medial calcification. MATERIALS AND METHODS: The mRNA expression of OPG and receptor activator of NF-kappaB ligand (RANKL) was measured in differentiated 3T3L1 adipocytes and adipose tissues. RESULTS: OPG mRNA expression increased with the differentiation of 3T3L1 adipocytes, while RANKL expression was not significantly altered. OPG mRNA was expressed at higher levels in white adipose tissue than in brown adipose tissue and was most abundant in the epididymal portion. In differentiated 3T3L1 adipocytes, Rosiglitazone and insulin reduced the OPG/RANKL expression ratio in a dose- and time- dependent manner. In contrast, tumor necrosis factor-alpha (TNF-alpha) increased the expression of both OPG and RANKL in a time-dependent manner. The OPG/RANKL ratio was at a maximum two hours after TNF-alpha treatment and then returned to control levels. Furthermore, OPG was abundantly secreted into the media after transfection of OPG cDNA with Phi C31 integrase into 3T3L1 cells. CONCLUSION: Our results indicate that OPG mRNA is expressed and regulated in the adipose tissue. Considering the role of OPG in obesity-associated inflammatory changes in adipose tissue and vessels, we speculate that OPG may have both a protective function against inflammation and anti-angiogenic effects on adipose tissue.

Keyword

Osteoprotegerin; receptor activator of NF-kappaB ligand; anti-angiogenic effects; obesity

MeSH Terms

3T3-L1 Cells
Adipocytes/cytology/drug effects/metabolism
Adipogenesis/genetics
Adipose Tissue/cytology/*metabolism
Animals
Cell Differentiation
*Gene Expression Regulation/drug effects
Hypoglycemic Agents/pharmacology
Insulin/pharmacology
Male
Mice
Osteoprotegerin/genetics/*metabolism
RANK Ligand/metabolism
Rats
Rats, Sprague-Dawley
Thiazolidinediones/pharmacology
Transfection
Tumor Necrosis Factor-alpha/pharmacology

Figure

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  Fig. 1 Expression of OPG/RANKL during adipocyte differentiation. Expression of OPG mRNA increased with the level of 3T3L1 preadipocyte differentiation, while mRNA levels of RANKL did not significantly change. OPG, osteoprotegerin; RANKL, receptor activator of NF-κB ligand. *p < 0.05.

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  Fig. 2 Differential expression of OPG/RANKL in various adipose depots in Sprague-Dawley rats. The pattern of mRNA expression of OPG/RANKL in subcutaneous adipose tissue was similar in mice, rats, and humans. Levels of OPG mRNA were higher in white adipose tissue than in brown adipose tissue, with the most abundant levels occurring in epididymal adipose tissue. WAT, white adipose tissue; BAT, brown adipose tissue; eWAT, epididymal white adipose tissue; pWAT, periomental white adipose tissue; sqWAT, subcutaneous white adipose tissue.

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  Fig. 3 Regulation of OPG and RANKL expression in the presence of TNF-α. TNF-α enhanced the expression of both OPG and RANKL mRNA in 3T3L1 preadipocytes. The OPG/RANKL ratio after TNF-α treatment was at a maximum two hours after treatment with TNF-α and then returned to control levels. *p < 0.05.

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  Fig. 4 Regulation of OPG/RANKL expression in the presence of insulin. Insulin (10 µg/mL) decreased the expression of OPG mRNA in 3T3L1 preadipocytes. *p < 0.01.

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  Fig. 5 Regulation of OPG/RANKL expression in the presence of Rosiglitazone. Rosiglitazone decreased the expression of OPG mRNA in 3T3L1 preadipocytes in a dose-dependent manner. *p < 0.05, **p < 0.01.

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  Fig. 6 OPG secretion after transfection of OPG cDNA into 3T3L1 preadipocytes. (A) Differentiation of 3T3L1 preadipocytes: a) 3T3L1 cells were first transfected with pcDNA 3.0-attB-OPG-IRES-puromycin DNA and pCMV integrase DNA for genomic integration. Then, the adipocytes were differentiated with puromycin selection for 14 days. b) 3T3L1 cells were first differentiated into adipocytes for 12 days and then transfected with pcDNA 3.0-attB-OPG-IRES-puromycin DNA only for acute transfection. The culture media was then collected and assayed 3 days after transfection. (B) Levels of OPG secretion as measured with an ELISA kit. Mock represents results from media obtained from 3T3L1 cells transfected with vector DNA. BD, before differentiation; AD, after differentiation; TBD, transfection before differentiation, which was from media obtained from 3T3L1 cells transfected with both pcDNA 3.0-attB-OPG-IRES-puromycin DNA and pCMV integrase DNA prior to differentiation into adipocytes; and TAD, transfection after differentiation, which was from media obtained from 3T3L1 cells differentiated into adipocytes first for 12 days and transfected with pcDNA 3.0-attB-OPG-IRES-puromycin DNA. Values are mean ± SE from 5 observations.

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