47NISR Research GRANT
2007 Research Grant

Basic and applied studies on functional factors in fermented
foods
Ryuichiro SATO
Department of Applied Biological Chemistry, The University of Tokyo

Research aims
Prevention of life style-related diseases with the use offood functions is highly required for the purpose of a reduction of healthcare cost in an aging society, Japan. Most life
style-related diseases are triggered by obesity occurring inassociation with disruption of lipid metabolism homeostasis. It is known that a variety of nuclear receptors are involved in the diseases. Forty-eight human nuclear receptors
are activated by inherent ligands bound to their C-terminal
domain, but some receptors are still referred to as orphan
receptors due to no ligands identified. Retinoic acid receptor-related orphan receptor a (RORa ) is one of orphan receptors, biological functions of which remain uncertain,
though cholesterol sulfate and its derivatives are speculated
to be endogenous specific ligands (1). We found that RORa
is expressed in adipocytes and that the activities of its
unidentified endogenous ligand elevate during adipogenesis. In this study we investigated biological functions ofRORa in adipocytes on the basis of the assumption thatfermented foods would contain a wide variety of steroid derivatives mimicking functions of RORa ligands.
Methods
Mouse 3T3-L1 preadipocyte cells were differentiatedinto adipocytes as previously described. A reporter plasmid
containing a GAL4 DNA-binding domain followed by theRORa ligand-binding domain (LBD) was constructed toevaluate the ligand-binding activity. The amounts of mRNAin adipocytes were determined by the real-time PCR
method. 3T3-L1 cells were infected with a lentivirus expression vector for either RORa or shRNA of RORa , andwere differentiated.
Results
Expression of RORa elevates during adipocyte differentiation. 3T3-L1 cells were differentiated into adipocytes andtotal RNA was recovered. The mRNA level of aP2, one ofdifferentiation markers, increased after day 4 after differentiation, and the mRNA level of RORa also similarly did up
to 5 times. Western blot analysis with specific antibodies
against RORa revealed that the protein level also elevated.
These results suggest that RORa regulates the expressionof its target genes during adipogenesis.

The RORa ligand activity was found in the culture mediumof adipocytes. Because adipocytes accumulate lipid dropletsin their cytosol during adipogenesis, it is postulated that thecontent of lipid soluble ligands of nuclear receptors wouldincrease. The expression of PPARg , a master regulator ofadipogenesis, elevates in the early stage of differentiation inassociation with the increase in its endogenous ligand,thereby being activated. It has been reported that theunidentified PPARg ligand is secreted in the culture
medium of adipocytes (2). Luciferase assays were carriedout using the RORa reporter gene together with the PPARg
reporter gene as a positive control. When the cells transfected with one of the reporter genes were incubated with aculture medium for day 6 differentiated adipocytes, theRORa ligand activity was detected in the culture medium
as well as the PPARg ligand activity. We failed to find any
activities for some other nuclear receptors tested in the assays. Taken together, it is suggested that the increased
RORa during adipogenesis in association with the increased particular ligands would regulate its target gene expression in adipocytes.
Overexpression of RORa restrains adipogenesis. RORa
was overexpressed about 10-fold in 3T3-L1 cells with thelentivirus expression vector, then the cells were differentiated. The overexpression decreased lipid droplets inadipocytes and down-regulated the expression of differentiation-related genes. In particular, the expression of perilipin, a protein located on the surface of lipid droplets andessential for lipid droplet formation, was reduced byRORa . As a result, it is likely that the lipid droplet formation was repressed by the function of RORa (3).
RORa knowkdown stimulates adipogenesis. The endogenous RORa level in 3T3-L1 cells was reduced down to lessthan 30% with the RORa shRNA expression lentivirus vector. When the cells were differentiated, more lipid droplets
were formed and the expression of differentiation-related
genes was induced. These results opposed to those observed in the RORa overexpressing cells clearly indicatethat endogenous RORa has an inhibitory effect on adipogenesis.
Conclusion
It is thought that PPARg , a master regulator of adipogenesis, stimulates differentiation of adipocytes through the48

increased its expression in the early and middle stage ofdifferentiation, and the elevated amount of its ligand.
RORa intensively studied in the current report behaved
quite similarly to PPARg during adipogenesis, and wasspeculated to play a pivotal role in adipocytes. Indeed, its
overexpression depressed adipogenesis, while its knockdown stimulated it. The fact that the expression of perilipin,
a lipid droplet surface protein essential for its formation, isparticularly downregulated by RORa raises the possibility
that RORa would have a kind of anti-obesity function. Although we tried to construct a reliable assay system to evaluate the RORa ligand activity, the assay using some fermented food factors was unable to be performed due to nopotent synthetic ligand as a positive control. The RORa lig-and activity found in the culture medium of adipocytes inthis report was only 2- to 3 - fold compared to that in thecontrol medium. The more sensitive assay system should beestablished by the use of endogenous or synthetic potent
ligand that will be found in the near future.
References1)

2)

Kallen, J., Schlaeppi, J-M., Bitsch, F., Delhon, I. and Fournier, B.
Crystal structure of the human RORa ligand binding domain incomplex with cholesterol sulfate at 2.2 Å., J. Biol. Chem. 279,14033–14038 (2004)
Kim, J. B., Wright, H. M., Wright, M. and Spiegelman, B. M.
ADD1/SREBP1 activates PPARg through the production of endogenous ligand. Proc. Natl. Acad. Sci. USA 95, 4333–4337 (1998)