Effect of Some Phytoestrogens on Metabolism of Rat Adipocytes

Effect of some phytoestrogens on metabolism ofrat adipocytes Katarzyna Kandulska, Leszek Nogowski, Tomasz Szkudelski

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Katarzyna Kandulska, Leszek Nogowski, Tomasz Szkudelski. Effect of some phytoestrogens on metabolism of rat adipocytes. Reproduction Nutrition Development, EDP Sciences, 1999, 39 (4), pp.497-501. hal-00900318

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Effect of some phytoestrogens on metabolism of rat adipocytes

Katarzyna Kandulska Leszek Nogowski Tomasz Szkudelski

Department of Animal Physiology and Biochemistry, University of Agriculture, Wolynska 35, 60-637 Poznan, Poland

(Received I July 1998; accepted 10 May 1999)

Abstract - The aim of this experiment was to evaluate the direct effect of genistein, daidzein and zearalenone on basal and hormone-induced lipogenesis and lipolysis in isolated rat adipocytes. In lipogenesis, daidzein and zearalenone were used at concentrations of 0.01, 0.1 and 1 mmol-L-1 and genistein at concentrations of 0.01, 0.3, 0.6 and 1 mmol-L-1. In lipolysis, concentrations of tested compounds were 0.01, 0.1 and I mmol-L-1. All tested compounds clearly inhibited basal and insulin (1 nmol-L-1) stimulated lipogenesis. Basal lipolysis was particularly enhanced by genistein and daidzein at its higher concentrations. The ability of zearalenone to potentiate of basal lipolysis was less marked. Epinephrine ( 1 Itmol!L-’)-stimulated lipolysis was inhibited by genistein at 1 mmol-L-1. At a concentration of 0.1 1 mmol-L-1 daidzein also augmented epinephrine-stimulated lipolysis and at its highest concentration exhibited an inhibitory effect, similar to genistein. Zearalenone reduced stimulated lipolysis, particularly at the highest concentration. © Inra/Elsevier, Paris. phytoestrogen / adipocyte metabolism

Résumé ― Effet de quelques phytoestrogènes sur le métabolisme des adipocytes du rat. Le but de ce travail était d’examiner les effets directs de la génistéine, de la daidzéine et de la zéaralé- none sur la lipolyse et la lipogenèse basale et après stimulation par l’insuline ou l’adrénaline sur des adipocytes isolés de rat. Dans le cas de la lipogenèse, la daidzéine et la zéaralénone ont été ajoutées aux concentrations de 0,01, 0,1 et 1 mmol.L-’ et la génistéine aux concentrations de 0,01, 0,3, 0,6 et 1 mmol-L-’. Dans le cas de la lipogenèse, les concentrations testées étaient les mêmes pour chaque composé : 0,01, 0,1 et 1 mmol.L-1. Tous les composés éxaminés réduisaient nettement la lipoge- nèse basale ou après stimulation par l’insuline (1 nmol!L-’). La lipolyse basale était particulière- ment augmentée par la génistéine et la daidzéine à leur plus forte concentration. L’efficacité de la zéa- ralénone sur la lipolyse basale était plus faible. La lipolyse après stimulation par l’adrénaline (1 ltmol!L-’) a été diminuée par la génistéine à sa plus forte concentration. La daidzéine (0,1 pmol.L-1) augmentait aussi la lipolyse. À sa plus forte concentration, la daidzéine a exercé un effet contraire simi- laire à celui de la génistéine. La zéaralénone diminuait la lipolyse, en particulier à forte concentration. @ Inra/Elsevier, Paris. phytoestrogène / métabolisme de l’adipocyte

* Correspondence and reprints E-mail: [email protected] 1. INTRODUCTION buffer pH 7.4, containing 3 mmol-L-1 glucose, 3 % bovine serum albumin (fraction V), mmol-L-1 HEPES and 2 Recently, a great deal of interest has been 10 mg.mL-1 collage- directed towards the findings that certain nase (from Clostridium histolyticum, type II). Incubation was for 90 min food exhibit activ- performed by shaking compounds estrogen-like at 37 °C. After incubation, the cells were rinsed are in con- ity. They present plants regularly four times with warm (37 °C) collagenase free sumed by animals and man, for instance Krebs-Ringer buffer and then filtered through soy, oat, barley, rye, wheat, corn, alfalfa, nylon mesh. Adipocyte counts were performed clover and others [9]. Because of their ori- using a microscope with a Burker-Turk count- chamber. gin, they are called phytoestrogens. The ing principal phytoestrogens are genistein, biochanin cou- daidzein, A, formononetin, 2.2. Lipogenesis in adipocytes mestrol and mycoestrogen-zearalenone which is Fusaria, a common produced by Fat cell suspensions (106 cells.mL-1) were field which also in organism proliferates incubated in plastic tubes at 37 °C with Krebs- poorly stored grains, oil seeds and hay [9]. Ringer buffer, pH 7.4, containing 3 % BSA, The exposure to these compounds can 10 mmol-L-1 HEPES, 0.5 pci of [U-14C] glu- adversely affect some physiological func- cose, 5.56 mmol-L-1 unlabelled glucose in the absence or of 1 nM insulin. Daidzein tions. It was observed that they are able to presence and zearalenone were used at concentrations of cause hyperestrogenic effects including uter- 0.01, 0.1 and 1 mmol-L-1, and genistein at con- ine enlargement in immature animals, estrus centrations of 0.01, 0.3, 0.6 and 1 mmol!L-’. The abnormalities and cycle fertility problems final volume was adjusted to 1 mL. Incubations [4]. Phytoestrogens were also found to par- were carried out with shaking for 90 min at ticipate in other events, e.g. cancer devel- 37 °C. The reaction was stopped by adding 5 mL opment [12], prevention of osteoporosis [2, of Dole’s extraction, containing isopropanol-hep- 3], corticosteroid disturbances, tane-1 N H2so4 (40:10:1). Tubes were shaken synthesis and then 2 mL of and 3 mL of were reduction of cardiovascular diseases [2], and H20 heptane added for lipid extraction. After shaking, sam- the of LDL oxidative protection against ples of the upper phase were transferred into modifications [11, 14]. There are also some counting vials containing a scintilation cocktail reports about their influence on lipid (OptiPhase ’Hi Safe’ Wallac) and total lipid metabolism; however, this effect is not well radioactivity was determined. known. The purpose of this work was to ascer- 2.3. Lipolysis in adipocytes tain the direct effect of some phytoestrogens (genistein, daidzein and zearalenone) Adipocytes (about 106 cells.mL-1) were incu- on lipid metabolism in isolated rat bated with shaking in plastic tubes at 37 °C for adipocytes. ’ 90 min with the Krebs-Ringer buffer containing 3 mmol-L-1 glucose, 10 mmol-L-’ HEPES and 3 % BSA in the absence or presence of 1 gmol-L-1 epinephrine. Phytoestrogens were used at con- 2. MATERIALS AND METHODS centrations of 0.01, 0.1 and 1 mmol-L-1. The final volume was adjusted to 1 mL. The glycerol 2.1. Preparation of adipocytes released from adipocytes, reflecting the inten- sity of lipolysis, was measured using the Male Wistar rats weighing 160 ± 5 g and kept Boehringer Mannheim test. Insulin (porcine, in standard conditions, were decapitated. Epi- monocomponent) was from NOVO, Nordisk, didymal fat tissue was pooled and adipocytes [U-14C] glucose was from New England Nuclear, were isolated according to the Rodbell method zearalenone from Kodak and genistein from [10] with minor modifications. The tissue was Fluka. All other reagents were purchased from rinsed with 0.8 % NaCI, cut into pieces and trans- Sigma. Results were statistically evaluated using ferred into a plastic flask with Krebs-Ringer one-way analysis of variance. 3. RESULTS strogens in the absence and presence of epinephrine. Genistein showed a dose- 3.1. Effect of phytoestrogens dependent enhanced effect on basal lipoly- on lipogenesis sis. Daidzein also stimulated basal lipolysis, but only at its higher concentrations. Table I shows glucose conversion to Zearalenone showed a less marked activa- lipids in adipocytes exposed to phytoestro- tion of basal lipolysis. Epinephrine-stimu- gens in the absence and presence of insulin. lated lipolysis was also influenced by genis- We observed that genistein clearly inhib- tein, but only at lower concentrations; at its ited basal lipogenesis in a concentration- highest concentration it inhibited this pro- dependent manner. A similar inhibition was cess. 0.1 1 mmol-L-1 daidzein also increased observed for both daidzein and zearalenone. lipolysis stimulation. At its highest concen- Insulin-stimulated lipogenesis was also tration it exhibited an opposite effect, sim- strongly reduced by phytoestrogens at all ilar to genistein. Mycoestrogen-zearalenone tested concentrations. reduced stimulated lipolysis, particularly at its highest concentration. 3.2. Effect of phytoestrogens on lipolysis 4. DISCUSSION

The phytoestrogens also significantly The performed experiments demonstrate affected lipolysis. Table II shows glycerol significant effect of genistein, daidzein and release from adipocytes exposed to phytoe- zearalenone on lipogenesis and lipolysis in isolated rat adipocytes. It was observed that glucose transporter GLUT4 [13]. Thus, all these compounds strongly inhibited lipo- restriction of insulin action and reduction genesis and enhanced basal lipolysis. The of glucose transport and metabolism seem to influence of phytoestrogens on cells is as be the most probable ways of inhibitory yet, poorly understood. Genistein action was action of tested compounds on lipogenesis in found as the most characterized phytoe- isolated rat adipocytes. strogen in the literature. In other experi- Lipolysis enhanced by genistein, daidzein ments used in similar concentra- genistein and zearalenone observed in our experi- tions was found to inhibit certain responses ments may be, particularly in the case of to insulin in adipocytes [1]. One of the effects genistein, the result of the inhibition of low is the reduction of the inhibitory action of Km cAMP phosphodiesterase activity. This insulin on An lipolysis [1]. inhibitory effect effect may cause fat mobilization as Kup- on both basal and insulin-stimulated lipo- pusamy and Das [6] reported for genistein genesis of genistein and other tested com- and a few other flavonoids. It was also pounds was observed in our experiments. observed that phytoestrogens act through This inhibitory effect may be expressed estrogen receptors [5] and may exhibit estro- either by the reduction of insulin action or in gen-like activity. Thus, phytoestrogens may, other ways. 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