SHORT COMMUNICATION

Stimulation of Lipolysis by Pycnogenol

Noboru HasegawaDepartment of Food and Nutrition, Nagoya Bunri College, Nagoya, Japan

We studied the influence of pycnogenol on the lipolysis of 3T3 L1 cells after differentiation. When pycno-genol or epinephrine was exposed to mature adipocytes, the smaller (less than 20mm2) intracytoplasmiclipid droplets selectively disappeared. These data suggest that pycnogenol stimulates lipolysis. Copyright# 1999 John Wiley & Sons, Ltd.

Keywords:3T3 L1 cell; pycnogenol; insulin; epinephrine; lipogenesis; lipolysis.

INTRODUCTION

Pycnogenol is a mixture of bioflavonoids with antiox-idative activity and is isolated from the bark of the Frenchmarine pine,Pinus pinaster(Masquelieret al., 1979). Itcontains a mixture of water soluble procyanidins, taxi-folin and phenolcarbonic acids. It controls the immunesystem (Cheshieret al., 1996), protects vascularendothelial cells (Ronget al., 1994–5) and reducesoedema or inflammation (Kuttanet al., 1981; Tixeret al.,1984) by radical-scavenging activity. However, nothingis known about its effects on obesity.

3T3 L1-preadipocytes are converted to adipocytes byinsulin (Green and Kehinde, 1975), and the intracellulardroplets of adipocytes are broken down by norepine-phrine (Chernicket al., 1986). In this study, pycnogenolwas tested for its lipolytic effects on the dispersion oflipid droplets by measuring the area of the droplets.

MATERIALS AND METHODS

Chemicals.Pycnogenol was provided by Tradepia Co.(Kasukabe, Saitama, Japan). Dulbecco’s modifiedEagle’s medium (DMEM) and DMEM:Ham F-12 (1:1)were purchased from Dainihon Pharmaceutical Co.(Tokyo, Japan). Epinephrine was from Daiichi Pharma-ceutical Co. (Tokyo, Japan). Bovine insulin, calf serum(CS) and fetal calf serum (FCS) were from JRHBiosciences (Lenexa, KS).

Cell culture. 3T3 L1-preadipocytes (American TypeCulture Collection, Rockville, MD) were grown inDMEM containing 10% CS, 100 U/mL penicillin and10 mg/mL streptomycin, at 37°C in humidified 5% CO2.

At confluence (day 0), the medium was changed toDMEM:F-12 (1:1) containing 10% FCS, and differentia-

tion was induced with 5 mg/mL insulin. The extent ofdifferentiation was quantified by staining intracellular oildroplets with Oil Red O after fixation with 5%formaldehyde.

On day 14 of culture, insulin was removed, and2� 10ÿ6 M epinephrine or 50mg/mL pycnogenol wasadded to the medium. The size of oil droplets wascarefully checked.

Data analyses.The phase-contrast micrographs wererecorded on a PC-AT using a CCD camera (Ikegami,Tokyo) and interface board (TARGA�, Truevision Inc.,IN). The area of droplets was determined using a JAVAsoftware (Jandel Scientific, CA).

Figure 1. Histogram of intracellular droplet area after 14 daysof pycnogenol or epinephrine treatment. 643, 341 or 288droplets in the control, pycnogenol or epinephrine, respec-tively, were counted.

PHYTOTHERAPY RESEARCHPhytother. Res.13, 619–620 (1999)

CCC 0951–418X/99/070619–02 $17.50Copyright# 1999 John Wiley & Sons, Ltd.

* Correspondence to: N. Hasegawa, Department of Food and Nutrition,Nagoya Bunri College, 2-1 Sasazuka-cho, Nishi-ku, Nagoya 451-0077, Japan.E-mail: hsgwn@nagoya-bunri.ac.jpContract/grant sponsor: Elizabeth Arnold Fuji Foundation.

Accepted 18 November 1998

RESULTS AND DISCUSSION

Administration of pycnogenol stimulated lipolysismainly in small droplets,and the cells seemedto havelost their cytoplasmicexpansion.Similar results wereobtainedin epinephrinecontainingmedium.When thedropletareawasexpressedasahistogram,thenumberofsmall droplets (<10mm2 or 10–20mm2) markedlydecreased(Fig. 1).b3 adrenoceptorsin adipocytesactivatecAMP accu-

mulation via G-protein (Fain and Garcia-Sainz,1983Gokmen-Poler et al., 1996), and intracellular lipiddroplets were broken down into small droplets bynorepinephrine(Sugiharaet al., 1987). Flavonoidscanhelp regulatethe productionof the cAMP phosphodies-terasein plateletor culturedcells and the incrementofcAMP causingthe effectson targetcells (Beretzet al.,

1982;MucsiandPragai,1985).Thesefindingssupportedtheideathatthelipolytic effectof pycnogenolwasduetothesameintracellularpathwayaswhenepinephrinewasapplied. Until now, the lipolytic effect has not beendemonstrated.In this report,the lipolytic effectoccurredselectivelyin small droplets.

In summary,this in vitro studyshowsthatthelypolyticeffectof pycnogenolwasmediatedvia thecontrolof thesecondmessenger.In additionto somebeneficaleffects,pycnogenolcanalsobeusefulin preventingobesityandmaintainingoptimal health.

Acknowledgements

This work was supported in part by the Elizabeth Arnold FujiFoundation.

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Copyright# 1999JohnWiley & Sons,Ltd. Phytother.Res.13, 619–620(1999)