Full text of DOI:10.1078/0944-7113-00148

Phytomedicine 9: 338–345, 2002
© Urban & Fischer Verlag
http://www.urbanfischer.de/journals/phytomed
Phytomedicine
Daidzein, coumestrol and zearalenone affect
lipogenesis and lipolysis in rat adipocytes
K. Szkudelska, T. Szkudelski and L. Nogowski
/
Department of Animal Physiology and Biochemistry, University of Agriculture, Wolyn´ska Poznan, Poland
Summary
Daidzein, coumestrol and zearalenone – compounds called phytoestrogens, considered as active bi-
ological factors affecting many important physiological and biochemical processes appeared to be
also significant regulators of adipocyte metabolism.
In our experiments the influence of daidzein (0.01, 0.1 and 1 mM), coumestrol (0.001, 0.01 and
0.1 mM), zearalenone (0.01, 0.1 and 1 mM) and estradiol (0.01, 0.1 and 1 mM) on basal and in-
sulin-stimulated (1 nM) lipogenesis from glucose and acetate was tested in adipocytes isolated
from growing (160 ± 5 g b.w) male Wistar rats. All tested compounds significantly attenuated
glucose conversion to lipids. In the case of daidzein and coumestrol, this effect was probably due
to inhibition of glycolysis.
Daidzein (0.01, 0.1 and 1 mM), coumestrol (0.01 and 0.1 mM) and zearalenone (0.01, 0.1 and 1
mM) affected also basal and epinephrine-stimulated (1 µM) lipolysis. Daidzein (0.01 and 1 mM)
augmented basal glycerides breakdown in adipocytes. The epinephrine-induced lipolysis was de-
pendent on daidzein concentration and its stimulatory (0.1 mM) or inhibitory (1 mM) influence
was observed. Zearalenone changed lipolysis only at the concentration of 1 mM and its effect was
contradictory in the absence or presence of epinephrine (the stimulatory or inhibitory effect, re-
spectively). Results obtained in experiments with inhibitors (insulin, 1 nM and H-89, 50 µM) and
activators (dibutyryl-cAMP, 1 mM and forskolin, 1 µM) of lipolysis allowed us to assume that
daidzein augmented basal lipolysis acting on PKA activity. The inhibitory effect of daidzein and
zearalenone on epinephrine-induced lipolysis is probably due to restriction of HSL action. The in-
fluence of coumestrol on glycerides breakdown was less marked. Estradiol augmented only
epinephrine-stimulated lipolysis.
Key words: daidzein, coumestrol, zearalenone, adipocytes, lipogenesis, lipolysis
᭿ Introduction
Phytoestrogens, naturally occurring diphenolic com-
pounds from plants and moulds, demonstrate the impor-
tant biological significance when consumed by humans
and animals. Many of their effects are considered as
health protective, although some deleterious ones are
documented as well. Phytoestrogens can bind to estro-
gen receptor (Shutt and Cox, 1972, Kuiper et al., 1998,
Molteni et al., 1995) so they may exert estrogen and
antiestrogen effects in organisms. Many of them caused
fertility problems, e.g. uterine hypertrophy or influence
on sexual differentiation and sexual behaviour (Adler-
creutz et al., 1995, Millington et al., 1964, Shutt and
Cox 1972, Bickoff et al., 1962, Tansey et al., 1998,
Gallo et al., 1999, Golden et al., 1998). However, sever-
al effects of these compounds are independent of their
interaction with estrogen receptor. It was demonstrated
that phytoestrogens could be effective in preventing
bone loss and some other postmenopausal problems
(Dodge et al., 1996, Tsutsumi 1995, Ishimi et al., 1999,
Fanti et al., 1998). A number of researchers also main-
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Daidzein, coumestrol and zearalenone affect
339
tain that they reduce cancer risk by protecting against collagenase free Krebs-Ringer buffer, filtered through
breast, colon and prostate cancer (Martin et al., 1978, nylon mesh and counted under the microscope with a
Lamartiniere et al., 1995 a, b, Wang and Kurzer, 1997, Bürker-Türk counting chamber. Cells viability was
Barnes, 1998, Adlercreutz et al., 1995, Barnes 1995, about 95% as determined by trypan blue exclusion.
Messina et al., 1994). Phytoestrogens inhibit aromatase
activity (Adlercreutz et al., 1993, Wang et al., 1994, Lipogenesis
Campbell and Kurzer, 1993) and, in this way, may de- The incorporation of glucose into lipids in adipocytes
crease risk of estrogen-dependent cancers. They are was studied in plastic tubes at 37 °C with Krebs-Ringer
also able to affect the immunological system – in nutri- buffer, pH 7.4, containing 3% BSA, 10 mM HEPES,
tionally relevant concentrations they enhance activation 0.5 µCi of [U14–C]glucose per ml (specific activity 9.80
of NK cells (Whalen and Green, 1998). The effect of Gbq/mmol, New England Nuclear Research Products),
phytoestrogens on cancer can be, however, different de- 5.56 mM unlabelled glucose in the absence or presence
pending on their concentration and stimulatory influ- of 1 nM insulin (porcine, monocomponent, NOVO,
ence on cancer growth was also observed (Wang et al., Nordisk). The incorporation of acetate into lipids was
1996). The advantageous influence of phytoestrogens evaluated in similar conditions, but 1 µCi of [14C]acetate
on the organism is also demonstrated by their ability to per ml (specific activity 0.832 GBq/mmol, POLATOM,
reduce LDLand VLDL cholesterol and increase HDL
Poland) was used instead of [U14–C]glucose and 10 mM
cholesterol (Anthony et al., 1996). This effect and the of unlabelled acetate was added. Daidzein (Sigma),
antioxidative properties (Mitchell et al., 1998) are re- zearalenone (Sigma) or 17 β-estradiol (Sigma) were
sponsible for prevention of heart diseases.
added at concentrations of 0.01, 0.1 and 1 mM. Coume-
Among different effects of plant estrogens observed strol (Fluka) was added at concentrations of 0.001, 0.01
in animals, their influence on lipid metabolism was and 0.1 mM. It was impossible to test higher concentra-
demonstrated (Nogowski, 1990, 1993, Nogowski et al., tion of coumestrol (1 mM), because it influenced pH of
1998). It was observed that phytoestrogens affected the incubation buffer. Each treatment was performed in
lipid metabolism in the liver and skeletal muscles and six replications, i.e. in six tubes incubated simultaneous-
changed some lipid parameters in plasma (Nogowski, ly and was repeated in two separate experiments (n =
1999). Alterations in the whole body lipid metabolism 12). Phytoestrogens were dissolved in dimethylsulfoxid
result also, at least in great part, from changes in (DMSO, Merck) and 10 µl of this solution (or 10 µl of
adipocytes. Indeed, in our previous experiments we DMSO in control tubes) was added to the buffer. The
demonstrated that genistein, daidzein and zearlaenone final volume in each tube was adjusted to 1 ml and 106
affect lipogenesis and lipolysis in the cells of white adi- cells were placed in this volume. Incubations were car-
pose tissue (Kandulska et al., 1999). However, the path- ried out with shaking. After 90 min the reaction was ter-
ways of action in these cells were investigated only in minated by adding 5 ml of Dole’s extraction mixture,
the case of genistein (Szkudelska et al., 2000).
containing isopropanol-heptane-1N H2SO4 (40:10:1)
The aim of this experiment was to determine the (Dole and Meinertz 1960). For the extraction of the
plausible mechanisms of daidzein, coumestrol and lipids 3 ml of heptane and 2 ml of H2O were added and
zearalenone effects on lipogenesis and lipolysis in iso- tubes were shaken. Samples of the upper phase were
lated rat adipocytes.
transferred into counting vials containing scintillation
coctail (OptiPhase ‘Hi Safe’ Wallac) and used for the
measurement of 14C incorporation into total lipids.
᭿ Materials and Methods
Lipolysis
Preparation of adipocytes
Fat cells suspensions (106 cells/ml) were incubated in
Male Wistar rats weighing 160 ± 5 g, kept in standard plastic tubes for 90 min by shaking at 37 °C with Krebs-
conditions in an air-conditioned room and at the con- Ringer buffer, pH 7.4 containing 3 mM glucose, 3%
stant temperature (22 ± 1 °C) were used in the experi- BSA, 10 mM HEPES. Phytoestrogens and estradiol
ment. Animals were fed ad libitum a standard laboratory were tested at the following concentrations: daidzein,
diet. Adipocytes were isolated according to the Rodbell zearalenone and estradiol – 0.01, 0.1 and 1 mM; coume-
method (1964) with minor modifications. After decapi- strol – 0.01 and 0.1 mM. The some part of experiment
tation epididymal fat pads were removed, rinsed and was performed in the presence of lipolytic activators –
placed for 90 min in Krebs-Ringer buffer, pH 7.4, 37 °C epinephrine (1 µM, with all phytoestrogens and with
containing 3 mM glucose, 3% bovine serum albumin estradiol), forskolin (1 µM, with daidzein and zear-
(BSA, fraction V), 10 mM HEPES and 2 mg/ml collage- alenone) or dibutyryl-cAMP (N6,2’-O-dibutyryladeno-
nase (EC 3.4.24.3, from Clostridium histolyticum, type sine 3’:5’-cyclic monophosphate, 1 mM, with daidzein
II). Isolated adipocytes were rinsed with warm (37 °C) and zearalenone) or lipolytic inhibitors – insulin (1 nM,

340
K. Szkudelska et al.
used instead of [U-14C]glucose daidzein did not affect
lipogenesis (Fig. 2). The highest (1 mM) concentration
of daidzein caused restriction in insulin-stimulated ac-
etate conversion to lipids (Fig. 2).
Coumestrol at all tested concentrations significantly
limited basal lipogenesis from glucose (Fig. 1). How-
ever, there was no effect of this phytoestrogen on glu-
cose conversion to lipids stimulated by insulin (Fig. 1).
Coumestrol appeared also to have no influence on the
basal and insulin-stimulated incorporation of [14C]ac-
etate into lipids (Fig. 2).
with daidzein) or H-89 (N-[2-(p-bromocinnamy-
lamino)ethyl]-5-isoquinolinesul-fonamide, 50 µM, ICN
Pharmaceuticals with daidzein). The final volume in
each tube was adjusted to 1 ml. The intensity of lipolysis
was measured by quantity of glycerol released from
adipocytes according to Foster and Dunn (1973). Re-
sults were statistically evaluated using one-way analysis
of variance (ANOVA) and multiple range test (p 0.05).
᭿ Results
Zearalenone strongly decreased lipogenesis from
glucose at all tested concentrations (Fig. 1). The similar
inhibition evoked by this mould estrogen was observed
in the presence of insulin (Fig. 1).
The effect of zearalenone on basal acetate conver-
sion to lipids was less marked - only at the highest con-
centration (1 mM) it decreased this process (Fig. 2).
Zearalenone at concentrations of 0.01 and 0.1 abated
Lipogenesis
The basal [U-14C]glucose conversion to lipids in
adipocytes incubated with daidzein was inhibited at its
higher concentrations (0.1 and 1 mM) (Fig. 1).
Daidzein at all tested concentrations exhibited also a
clear inhibitory effect on lipogenesis from glucose in
the presence of insulin (Fig. 1). When [14C]acetate was
Fig. 1. The effect of daidzein, coumestrol, zearalenone and
estradiol on basal (upper plot) and insulin-stimulated (1 nM,
lower plot) lipogenesis from glucose in isolated rat
adipocytes. Values are means ± SEM from two separate ex-
periments, n = 6 in each experiment; A, B, C, D – differences
statistically significant (p 0.05) between different phytoe-
strogen concentrations.
Fig. 2. The effect of daidzein, coumestrol, and estradiol on
basal (upper plot) and insulin-stimulated (1 nM, lower plot)
lipogenesis from acetate in isolated rat adipocytes. Values are
means ± SEM from two separate experiments, n = 6 in each
experiment; A, B, C – differences statistically significant (p
0.05) between different phytoestrogen concentrations.

Daidzein, coumestrol and zearalenone affect
341
Zearalenone manifested significant lipolytic activity
in the absence of epinephrine only at the highest con-
centration (1 mM), but at the same concentration it re-
stricted epinephrine-induced lipolysis (Fig. 3).
Daidzein and zearalenone at the concentration of 1
mM decreased lipid breakdown caused by lipolytic ac-
tivators – forskolin and dibutyryl-cAMP (Fig. 5).
Lipolysis stimulated by daidzein at both tested con-
centrations – 0.1 and 1 mM was clearly diminished by
H-89, but insulin had no effect on this process (Fig. 4).
There were no significant changes in basal lipolysis
in the presence of estradiol (Fig. 3). However,
epinephrine-stimulated lipolysis was potentiaed by this
hormone at all tested concentrations (Fig. 3).
᭿ Discussion
Results obtained in this study clearly demonstrate that
daidzein, coumestrol and zearalenone affect lipogene-
sis and lipolysis in isolated rat adipocytes.
Daidzein restricted both basal and insulin-stimulated
glucose conversion to lipids (Fig. 1). This process is
preceded by glucose transport into cells and involves
its metabolism to pyruvate and glycerol-3-phosphate.
Pyruvate is transformed to acetyl-CoA and, finally,
malonyl-CoA. Then, long-chain fatty acids are formed
and are esterified to triglycerides. The inhibitory effect
of daidzein on basal glucose metabolism to lipids indi-
cates that this phytoestrogen restricts predominantly
insulin independent steps of lipogenesis. This observa-
tion is in agreement with literature data demonstrating
that daidzein is not an inhibitor of protein tyrosine ki-
nase (Vera et al., 1996) and may affect some processes
without influencing insulin receptor.
Daidzein at concentrations of 0.01 and 0.1 mM was,
however, without effect on lipogenesis when acetate
was used as a substrate for this process (Fig. 2). These
results indicate that daidzein restrains glucose conver-
sion to lipids by inhibiting only the initial, i.e. those
taking place before acetyl-CoA formation, steps of li-
pogenesis. Since Vera et al., (1996) demonstrated that
this phytoestrogen does not affect hexose transporter
GLUT1, the inhibitory action of daidzein on glucose
transport may be ruled out. Therefore, it can be stated
that daidzein attenuates lipogenesis via the restriction
of glucose metabolism to acetyl-CoA, whereas other
steps of this process are unaffected. However, at the
highest concentration tested (1 mM), daidzein restrict-
ed also final steps of lipogenesis, since its inhibitory
action on acetate metabolism was observed.
Fig. 3. The effect of daidzein, coumestrol, zearalenone and
estradiol on basal (upper plot) and epinephrine-stimulated
(1 µM, lower plot) lipolysis in isolated rat adipocytes. Values
are means ± SEM from two separate experiments, n = 6 in
each experiment; A, B, C – differences statistically signifi-
cant (p 0.05) between different phytoestrogen concentra-
tions.
insulin-induced lipogenesis from acetate, but it mani-
fested slight stimulatory effect at the highest concentra-
tion (1 mM) (Fig. 2).
Estradiol restricted basal lipogenesis from glucose
only at the highest concentration (1 mM). In conditions
with insulin, it inhibited this process also at 10-fold
lower concentration (Fig. 1). The effect of this sterol on
lipogenesis from acetate was negligible (Fig. 2).
Lipolysis
Daidzein evoked marked augmentation of basal lipoly-
sis at higher concentrations – 0.1 and 1 mM (Fig. 3). In
the presence of epinephrine daidzein increased glyc-
erol release from adipocytes at the concentration of 0.1
mM but at the highest concentration – 1mM – it exhib-
ited the inhibitory effect (Fig. 3).
There were no significant effects of coumestrol on
basal lipolysis but at the concentration of 0.1 mM a
stimulatory effect on epinephrine-induced process was
observed (Fig. 3).
Zearalenone, similarly to daidzein, significantly di-
minished basal and insulin-stimulated lipogenesis from
glucose (Fig. 1). Thus, it strongly affected lipogenesis
by inhibiting insulin independent steps of this process.

342
K. Szkudelska et al.
Fig. 5. The effect of daidzein (1 mM) and zearalenone (1
mM) on lipolysis stimulated by different lipolytic agents:
forskolin (F, 1 µM) or dibutyryl-cAMP (DB, 1 mM) in iso-
lated rat adipocytes. Values are means ±SEM from two sep-
arate experiments, n = 6 in each experiment; A, B - differ-
ences statistically significant (p 0.05) between lipolytic
agent with or without phytoestrogen.
Fig. 4. The effect of insulin (1 nM) or H-89 (50 µM) on
daidzein-induced (0.1 and 1 mM) lipolysis in isolated rat
adipocytes. Values are means ±SEM from two separate ex-
periments, n = 6 in each experiment; A, B - differences statis-
tically significant (p 0.05) between appropriate phytoestro-
gen concentration with or without the inhibitory agent.
Results obtained during incubations with acetate (Fig. at the highest concentrations restricted lipogenesis
2) indicate that its effect on the steps of lipogenesis from glucose, but did not affect acetate conversion to
after acetyl-CoA formation varies depending on its lipids (Fig. 1 and 2). These results demonstrate the in-
hibitory action of this hormone only at the processes
of lipogenesis occurring before the formation of
acetyl-CoA.
concentration and the absence or presence of insulin.
Coumestrol, at all tested concentrations, diminished
basal glucose metabolism to lipids (Fig. 1) but did not
affect lipogenesis from acetate (Fig. 2). Hence, its in-
hibitory action on lipogenesis involves only those steps
of this process which occur before the formation of
acetyl-CoA. It is noteworthy that the inhibitory effect
of coumestrol on lipogenesis from glucose was com-
pletely abolished in the presence of insulin. Therefore,
it is possible that this phytoestrogen restricts basal glu-
cose transport by GLUT1 whereas insulin, by stimulat-
ing glucose transport via GLUT4, may suppress this in-
hibitory effect. This assumption may be supported by
findings that coumestrol stimulated glucose conversion
to lipids in perfused rat liver. In hepatocytes glucose is
transported via GLUT2 and this transport was not in-
hibited by coumestrol (Nogowski 1999). However, the
effect of this phytoestrogen on glucose metabolism to
acetyl-CoA in adipocytes can not be excluded. It is
known that insulin potentiates several steps of glucose
conversion to acetyl-CoA and, thereby, may overcome
the inhibitory effect of coumestrol.
Phytoestrogens affecting lipogenesis appeared to
have some influence also on triglycerides breakdown.
Basal and epinephrine-induced lipolysis observed in
isolated adipocytes was significantly changed in the
presence of daidzein and zearalenone in the incubation
medium (Fig. 3). Upon epinephrine treatment, a lipoly-
tic cascade is activated. The main steps of this cascade
involve: activation of adenylate cyclase by ß-adrener-
gic receptors, rise in cAMP and activation of protein
kinase A (PKA) resulting in the stimulation of lipolysis
by hormone sensitive lipase (HSL).
Basal triglycerides breakdown was augmented by
daidzein at concentrations of 0.1 and 1 mM (Fig. 3).
Nichols and Morimoto (1999) demonstrated that this
phytoestrogen possesses a weak inhibitory effect on the
activity of low Km cAMP phosphodiesterase. This ef-
fect was found to be weaker than that of genistein, how-
ever, the inhibition of this enzyme by daidzein may aug-
ment cAMP content in adipocytes resulting in the stimu-
lation of lipolysis. To test this possibility adipocytes
were incubated with daidzein in the presence of insulin.
The effect of daidzein, coumestrol and zearalenone
on lipogenesis was compared with estradiol. Estradiol

Daidzein, coumestrol and zearalenone affect
343
Insulin is a strong anti-lipolytic factor exerting its ac- adipocytes even in the absence of lipolytic agents (Mori-
tion, at least in great part, by the activation of low Km moto et al., 2001) and that phosphatidylcholine (Tsujita
cAMP phosphodiesterase (Degerman et al., 1990; Eriks- et al., 1995) and perilipins (Clifford et al., 2000) present
son et al., 1995). The activation of this enzyme allows on the surface of lipid droplets prevent triglycerides
insulin to diminish lipolysis stimulated by agents in- breakdown under basal conditions. Perilipins and HSL
creasing the amount of cAMP in adipocytes. This hor- undergo translocation upon stimulation of lipolysis and
mone however is, unable to suppress basal lipolysis than HSL is able to hydrolyse triglycerides (Clifford et
(Morimoto et al., 1998). In our experiment, insulin al., 2000). Therefore, the diminution of lipolysis evoked
failed to attenuate lipolysis evoked by daidzein (Fig. 4). by daidzein and zearalenone may be caused by direct in-
It appears that this was not due to daidzein-induced sup- hibition of HSL activity or by interaction with factors
pression of insulin action in adipocytes. Such inhibitory regulating the lipolytic action of this enzyme.
effect on insulin action was demonstrated for genistein
but not for daidzein (Abler et al., 1992). The lack of the sis, but only in he presence of epinephrine (Fig. 3).
inhibitory effect of insulin indicates that the previously In this experiment and in experiments performed
Estradiol manifested the stimulatory effect on lipoly-
demonstrated (Nichols and Morimoto, 1999) influence previously (Kandulska et al., 1999) we observed that
of daidzein on cAMP phosphodiesterase activity was in- the influence of daidzein and zearalenone on lipolysis
sufficient to reveal its lipolytic activity. This is in agree- may be contradictive depending on their concentration
ment with results obtained by Kuppusamy and Das and depending on the presence or absence of
(1992) who demonstrated that not all flavonoids capable epinephrine. This contradictive effect is characteristic
of inhibiting cAMP phosphodiesterase induced lipolysis for different aspects of phytoestrogens action and was
in adipocytes. Thus, augmented lipolysis observed in the also observed in the case of lipolysis stimulated by
presence of daidzein was evoked by its action down- genistein (Szkudelska et al., 2000). This effect results
stream from adenylate cyclase. Its influence may in- probably from the ability of each daidzein, zearalenone
volve PKA or HSL. Incubation of adipocytes with H-89, and genistein to act at several steps of lipolysis.
a potent and specific inhibitor of PKA, abolished
Kuiper et al. (1998) showed that genistein, daidzein
daidzein-induced rise in triglycerides breakdown and zearalenone had lower estrogenic potency than
(Fig. 4). All these results indicate that daidzein may aug- estradiol. Daidzein and zearalenone had also more than
ment basal lipolysis by activation of PKA.
100 and 5 times lower binding affinity to the estrogen
The similar mechanism of action may be responsible receptor than estradiol, respectively. However, we ob-
for the stimulatory effect of this phytoestrogen at the served that the influence of these phytoestrogens on li-
concentration of 0.1 mM on epinephrine-induced lipol- pogenesis and lipolysis was greater in comparison to
ysis (Fig. 3). However, daidzein at the highest concen- this hormone. The binding affinity of coumestrol to the
tration (1 mM) exerted opposite effect and significantly estrogen receptor is similar to estradiol (Kuiper et al.,
limited epinephrine-induced triglycerides breakdown. 1998), but the influence of this phytoestrogen on lipoge-
To ascertain more precisely this action, lipolysis was nesis from glucose and on epinephrine-stimulated lipol-
stimulated by factors acting at different steps of the ysis was lower than estradiol. Genistein, which binds to
lipolytic cascade. The inhibitory effect of daidzein was the estrogen receptor with affinity lower than estradiol,
still observed when lipolysis was induced by direct ac- had the higher ability to affect adipocytes metabolism
tivation of adenylate cyclase (by forskolin) or PKA(by
(Szkudelska et al., 2000). It was also observed that phy-
dibutyryl-cAMP) (Fig. 5). These results allow us to toestrogens evoked several changes in adipocytes,
conclude that daidzein, at the concentration of 1 mM, which were not demonstrated for estradiol, such as inhi-
restricts epinephrine-induced lipolysis by diminution bition of cAMP phosphodiesterase (Nichols and Mori-
of the HSL action.
moto 1999). The comparison of the phytoestrogens and
Zearalenone affected lipolysis only at the highest con- estradiol influences on lipogenesis and lipolysis and
centration (1 mM, Fig. 3). Its action involved stimula- their binding affinity to the estrogen receptor indicate
tion of basal and inhibition of epinephrine-induced that the effects of daidzein, coumestrol and zearalenone
lipolysis. Triglycerides breakdown evoked by direct ac- on these processes may be independent of their estro-
tivation of adenylate cyclase or PKAwas also depressed
by zearalenone (Fig. 5). Thus, the inhibitory action of
genic activity.
In conclusion, our results revealed that daidzein,
this mycoestrogen on epinephrine-induced lipolysis coumestrol and zearalenone significantly attenuated li-
seems to be similar to that of daidzein, i.e. via inhibition pogenesis in isolated rat adipocytes. In the case of
of HSL action. A similar mechanism of action was pre- daidzein and coumestrol this effect was due to inhibi-
viously proposed for genistein (Szkudelska et al., 2000). tion of acetyl-CoA formation from glucose. The effect
The exact mechanism of this inhibition is difficult to as- of zearalenone was more complex, depending on its
certain. It was demonstrated that HSL is active in concentration and presence or absence of insulin.

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Katarzyna Szkudelska, University of Agriculture,
Wo lyn´ska 35, 60-637 Poznan, Poland
Department of Animal Physiology and Biochemistry,
Tel./Fax: ++48-61-84 87 196;
e-mail:tszkudel@jay.au.poznan.pl
/