SHORT COMMUNICATION

Inhibition of Lipogenesis by Pycnogenol

Noboru Hasegawa*Department of Food and Nutrition, Nagoya Bunri College, Nagoya, Japan

The influence of pycnogenol on the adipose conversion of 3T3-L1 cells by insulin was studied. In week 3of culture with insulin, pycnogenol was found to inhibit significantly the expression of glycerophosphatedehydrogenase (p< 0.01). This finding suggests that pycnogenol inhibits the accumulation of lipid dro-plets in adipose tissue. Copyright# 2000 John Wiley & Sons, Ltd.

Keywords:3T3-L1 cell; pycnogenol; insulin; lipogenesis; glycerophosphate dehydrogenase; adipose conversion.

INTRODUCTION

Pycnogenol is a mixture of bioflavonoids with antioxi-dative activity (Masqueleret al., 1979), which has beenused in European countries as a dietary supplement. Itenhances immune function (Liuet al., 1998), decreasesplatelet aggregation and inhibits the formation of low-density lipoprotein (Fitzpatricket al., 1998), andmodulates nitrogen monoxide metabolism (Virgilietal., 1998). In addition to these beneficial effects, we havesuggested that pycnogenol stimulates lipolysis in 3T3-L1cells (Hasegawa, 1999). However, nothing is knownabout its effects on adipose conversion and lipidaccumulation.

Cytosolic glycerophosphate dehydrogenase (GPDH) isencoded at two different loci, and the appearance of thestable ‘adult’ form is linked to changes characteristic ofadipose conversion (Wise and Green, 1979). In thepresent study, pycnogenol was tested for an antiadipo-genic effect on the adipose conversion of 3T3-L1preadipocytes by measuring the expression of GPDH.

MATERIALS AND METHODS

Chemicals. Pycnogenol was provided by TradepiaCo. (Kasukabe, Saitama, Japan). Dulbecco’s modifiedEagle’s medium (DMEM) and DMEM: Ham F-12 (1:1)were purchased from Dainihon Pharmaceutical Co. (Tokyo,Japan). Bovine insulin, calf serum (CS) and fetal calf serum(FCS) were from JRH Biosciences (Lunexa, KS).

Cell culture. 3T3-L1 preadipocytes were cultured by themethod previously described (Hasegawa, 1999). Briefly,cells were grown in DMEM containing 10% CS,100 U/mL penicillin and 10mg/mL streptomycin, at37°C in a humidified 5% CO2. At confluence (day 0), themedium was changed to DMEM:F-12 (1:1) containing

10% FCS, and differentiation was induced with 5 mg/mLinsulin.

Pycnogenol (50mg/mL) was present from the first dayof the culture.

Assays.Cells were removed 21 days after the differ-entiation process. For removal, cells were washed withcold PBS and scraped from the dishes in a prechilledbuffer consisting of 25 mM Tris hydrochloride buffer and1 mM EDTA (pH 7.5). The activity of GPDH wasmeasured spectrophotometrically in sonicated cell ex-tracts according to an established procedure (Wise andGreen, 1979). One unit of enzyme activity corresponds tothe oxidation of 1 nmol NADH /min per mg protein. Theprotein content of the homogenates was measured using a

Figure 1. Effect of pycnogenol on insulin-induced GPDHactivity in 3T3-L1 cells. Results are presented as the mean -� SE of four experiments in duplicate; **p< 0.01.

PHYTOTHERAPY RESEARCHPhytother. Res.14, 472–473 (2000)

Copyright# 2000 John Wiley & Sons, Ltd.

* Correspondence to: Dr 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.

Received 2 September 1999Accepted 21 November 1999

commercially available kit (Coomassie protein assayreagent, Pierce, IL). Bovine serum albumin was used as astandard.

Statistics. Results are expressed as the mean� SE.Student’st-test was used for statistical comparison.

RESULTS AND DISCUSSION

In week 3 of culture with insulin, the fat cells exhibitedlarger intracytoplasmic lipid droplets. During this period,GPDH activity, which was used as a marker ofdifferentiation, increased from undetectable levels tobetween 100 and 187 U/mg cellular protein (Fig. 1).When pycnogenol was added, lipogenesis due to insulinwas inhibited and a significant decrease in GPDH activitywas observed (Fig. 1).

Pycnogenol is known as a potent antioxidant (Rongetal., 1994–1995). Insulin activated NADPH-oxidase on aplasma membrane, and produced H2O2 accelerated

adipose conversion of 3T3-L1 preadipocytes (Kreiger-Brauer and Kather, 1992, 1995). H2O2 has beenimplicated as a second messenger (Ramasarma, 1982).Together with the above findings, it is proposed thatpycnogenol inhibits differentiation of adipocytes not bycompeting with the insulin receptor but by prevention offree radical mediated processes. When mature adipocyteswere exposed to pycnogenol, smaller intracytoplasmiclipid droplets selectively disappeared (Hasegawa, 1999).These results suggest that pycnogenol inhibits lipogen-esis and stimulates lipolysis.

In summary, this in vitro study shows that theantiadipogenic effect of pycnogenol is mediated via thecontrol of the second messenger. In addition to otherbeneficial effects, pycnogenol can also be useful inpreventing obesity and maintaining optimal health.

Acknowledgement

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

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Copyright# 2000 John Wiley & Sons, Ltd. Phytother. Res.14, 472–473 (2000)