300
Horm. Metab. Res. 9 (1977) 300-304
Effects of Clofibrate on the Human Fat Cell Adenylate Cyclase System
H. Kather, Gabriela Simon-Crisan, B. Vogt and B. Simon
Klinisches Institut für Herzinfarktforschung an der Medizinischen Universitätsklinik Heidelberg
(Dir. Prof. Dr. Dr. h.c. G. Schettler), Germany
Summary
The effects of chlofibrate on the adenylate cyclase system
of human adipoeytes were studied.
Oofibrate reduced basal as weil as hormone-(NaF)stimulated
adenylate cyclase activities to about the same extent (45%
inhibition at 1 mg/mi clofibrate).
The relative extent of hormonal stimulation was not altered
by this compound.
The inhibitory action of c10fibrate was non-competitive
with respect to the substrate ATP and cofactors (Mg2+-
ions).
Inhibition of enzyme activity was deteetable after 2.5 min.
Our results suggest that the antilipolytic aetivity of c10-
fibrate is mediated via inhibition of the catalytie subunit
of the fa t eell adenylate cyc1ase.
Key-Words: Human Adipocytes - Adenylate Cyc1ase Ac·
tivity - aofibrate - Lipolysis - Adrenaline
Introduction
Clofibrate (ethyl p-chlorophenoxyisobu tyrate) is
commonly used as a lipid lowering agent in hyper-
lipidemia (Fredn'ckson 1972, Havel and Kane 1973).
Among the numerous in vitro actions of this agent
an antilipolytic affect has been described (Barrett
1966, Barrett and Thorp 1968, Smigura, Farkas
and Angel 1973). This property has been suggested
to contribute to the hypolipidemic action of clo-
fibrate (D'Costa and Angel 1975).
From metabolic studies indirect evidence has been
presented that the antilipolytic activity of clofibrate
is media ted via inhibition of membrane associated
adenylate cyclase activity (E.C.4.6.1.1) (D'Costa
and Angel 1975). Direct effects of this drug on the
enzyme system, however, have not been clearly do-
cumented. We, the re fo re , studied the effect of cla-
fibrate on the activlty of the adenylate cyclase sys-
tem in human fat cell ghosts.
Methods
Source of biopsies
Biopsies of subcutaneous adipose tissue were obtained from
8 patients undergoing surgical treatment. The clinical eha-
raeteristies of the patients are shown in Table 1.
Parts of this work has been published as Short Communi-
eation in this Journal (Vol. 8: 246-247, 1976)
Reeeived: 5 June 1976 Accepted: I Oec. 1976
© Georg Thieme Verlag Stuttgart
No attempt was made to seleet the patients on the basis
of age, sex, weight or disease, except that eaeheetie persons
were excluded. 'Ille patients were opera ted after an over-
nigh t fast. Anesthesia was induced with a short acting bar-
biturate and maintained with halothan, nitrous oxyce and
oxygen. The biopsies were usually obtained after the skin
incision at the start of the operation.
Preparation of fat cell ghosts
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Adipose tissues were cut into 20-25 mg fragments and fat
ceUs were isolated essentially aecording to Rodbell (1964),
except that higher concentrations of eoUagenase (3 mg/mI)
and Tris-HCI buffer (0.025 M), pH 7.4, instead of Krebs-
Ringer-bicarbonate buffer were used.
Fat cell ghosts representing a plasma membrane-rich frac-
tion isolated from hypotonie Iysates of free fat eells were
prepared according to Pohl et al. (1971). The Iysing medium
contained 0.05 M MgCI 2 , 0.05 M ATP, 0.05 M CaCI 2 , 0.1
M KHC0 3 and Tris-HCI (0.01 Ml, pH 7.6. The ghosts were
suspended in 0.1 mM KHC0 3 in a final concentration of
0.25 to 2.5 mg protein/ml.
Adenylate cyc1ase activity
The adenylate eydase activity of fat cell ghosts was deter-
mined aceording to Salomon ct al. (1974). Thc ineubation
mixture contained 25 mM Tris-HCI, pH 8.5, 5 mM MgCI 2 ,
20 mM creatine phosphate, 100 V/mi ereatine phosph~
kinase, 1 mM cydic AMP, I mM (~32P) ATP (40-50 cpm/
pmole) and adrenaline (6.1 x 10 -4M), nor-adrenaline (6.1
x 10-4 M) or sodium fluoride (20 mM). The reaction was
initiated by the addition of membranes in a final reaction
volume of 100 ul to give 0,05-0..5 mg/mI of membrane pro-
tein. Temperature was 300 C, ineubation time 15 min.
Cydic (32P) AMP was purified by column ehromatography
using Dowex AG 50 W-X 4 and neutral alumina.
The protein content of the samplcs was dctermined accord-
ing to Lowr-y et al. (1951) using bovine serum albumin as
standard.
Sodium c10fibrate was dissolved in 0.1 N NaOH, the pH
of the solution was adjusled 10 pH 8.5 and directly added
10 the incubation media.
The experiments shown in Figures 1-4 were repeated at
least three times in order to ensure that Ihc qualitdtive pat-
tern of effects were reproducible.
Materials
(a- 32 P) ATP (2-6 Ci/mmole) and eydic (3H) AMP (27 Ci/
mmole) were purehased from the Radiochemical Centre
Amersham Bueks, V.K.
Sodium c10fibrate was obtained from ICI-Pharma, Plank-
stadt, adrenaline, noradrenaline and sodium fluoride from
Mer.::k AG, Darmstadt, FRG.
All other chemieals and reagents were of the highcst grade
commercially available.
 Effects of Clofibrate on the Human Fat CeJl Adenylate Cyclase System 301

Table 1. Clinical characteristics in the patients studied.

Sex Age Weight Height Disease % inhibition of adenylate cyclase

(kg) (cm) activity by 1 mg/mi clofibrate

79 81 171 inguinal hernia incarc. 42

73 90 165 gallstones, diabetes mell. 39
48 84 166 uterus myomatosus, hypertension 45
70 65 158 arteriaJ obstructive disease, hypertension 38
56 108 163 benign soft tissue tumor 52
54 71 159 gastric carcinoma 47
56 70 164 uterine cervix cancer 43
22 10 163 macromastia 46

9
~

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,
E
ll"I

S
- - NaF

~ 7 __ control
[ .......... .. clofibrate
~
'"8. 5
~ Cl
l:
:~ "<{
o---<l
u u
"<{'li"' N:Jradrenaline
~~ 3
" "0E
U
G ~

~
~
.!:! _Basal
..
~
"b
"<{ 0

Clofibrate [g/m/] ADRENALINE [M]

Fig. 1. Effect of increasing concentrations of clofibrate on Fig. 2. Effect of clofibrate on the adrenaliJle sensitivity of
b~sa1 (e--e), noradrenaline (0-0) as weil as NaF (.-.) the adenylate cyclase system in human fat cells. The extent
stimulated adenylate cyclase activity. For experimental de- of adrenaline stimulation is expressed relative to respective
tails see Methods. basal enzyme activity.

Results As seen in Figure I non-stimulated and hormone

(NaF) activated activities were inhibited to the same
Figure 1 shows the effect of various concentrations
degree at a different flXed concentration of clofibrate.
of clofibrate (0.01 to 1 mg/mI) on non-stimulated
This fmding is further illustrated in Figure 2 where
and hormone-(NaF)activated enzyme activities. In-
the stimulation of enzyme activity by various con-
creasing concentrations of this drug caused a dose-
centrations of adrenaline is expressed relative to the
dependent inhibition of all expressions of enzyme
respective non-stimulated (basal) enzyme activity.
activity (non-stimulated i.e. basal, noradrenaline- as
It is obvious that clofibrate though inhibiting the
weIl as NaF-stimulated activities) up to 45% at a
absolute rates of cAMP accumulation did not affect
clofibrate concentration exceeding the therapeutic
the hormone sensitivity of the enzyme system i.e.
levels by about 1 order of magnitude (0.1 mg/mI).
the relative extent of hormonal stimulation.
At a dose corresponding to therapeutic concentra-
tions in vivo the inhibition of enzyme activtty was The inhibitory action of clofibrate was detectable
about 15-20%. after 2.5 minutes. This is illustrated in Figure 3
showing the effects of clofibrate on the time course
The inhibitory action of ethyl-p-chlorophenoxyiso-
of non-stimulated enzyme activity. The rates of
butyrate® was confirmed in fat cell ghosts obtained
cAMP formation were linear up to 15 min, a 35-38%
from 8 patients suffering from various diseases (Ta-
inhibition occurred throughout in the presence of
ble 1).
clofibrate.
302 H. Kather, Gabriela Simon-Crisan, B. Vogt, B. Simon

........ Basat
1750 t:r---l:l • etof/brale

1500

!::
~
e
Cl.
1250

~
.
'-
Cl.
1000
•
11.
~

'",
l.>
750

,--,
4-

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'"
'--' 500
"0
E
Cl.
• Fig. 3. Time course study of
non-stimulated cAMP accumula-
tion by human fat cell ghosts
in the prcsence (/,~-j\,)or absence
5.0 10.0 15,0 20.0 I (min) (e-e) of c1ofibratc.

basal activity [MgCI2 lmM] adrenaline-stlmulafed acfivlfy

c:

2
3.

---
()----()
control
• c10fibrate
~
E 3.
"~c:
0
I...
Cl..
2.5

2.0
___ confrol

Ol
E:
I...
1.S 'Cl.." 1.S
It.
1.0
• ~
<:{ 1.
u
~
OS ~ 05
L..:-.J
"0
E
c:
aOl aOS 0.1 0,5 1,0 5,0 [ aql aoS al aS 1.0 S,o
Arp mM J
Fig. 4. Effect of ATP concentration on the inhibitory action of c10fibrate in the presence (right side) and absence (lcft
side) of adrenaline.
Mg2+ concentration was 1 mM.

Table 2 shows the inhibitory effect of clofibrate
(I mg/mI) on the enzyme system at two concentra-
tions of Mg 2 + (5 and 20 mM). The inhibition of
basal and hormone-stimulated enzyme activities was
about 45 to 47% at both Mg 2 +-concentrations in-
dicating that the action of clofibrate was not med-
iated by any chelation effect on Mg2 +.
Figure 4 illustrates the effect of I mg/mi c10fibrate
on basal (left side) and adrenaline-stimulated (right
side) enzyme activities at different concentration
of ATP (0.01 to 5 mM). The Mg 2 + concentration
was 1 mM in these experiments. Optimal enzyme ac-
tivities of controls and clofibrate-treated ghosts oc-
curred at a ATP:Mg2 t ratio of about 1: 1. ATP in
 Effects of Clofibrate on the Human Fat Cell Adenylate Cyclase System
Table 2. Effeet of Mg2+-ions on the inhibition of human fat cell adenylate cyclase by clofibrate
Additions Clofibrate
+ 0.64 ± 0.24
L-Adrenaline
(1 mM) + 1.17 ± 0.16
L-Noraruenaline
(1 mM) + 0.98 ± 0.17
Adenylate cyclase activity is expressed in nM [p32] cAMP liberated/mg protein x 15 min.
excess of Mg2+ was inhibitory in both enzyme pre-
parations. It is clear that addition of clofibrate re-
duced only the Vmax of basal and activated enzymic
reaction without influencing the apparent Km for the
substrate ATP. These results indicate that the inhi-
bition of the enzyme system by clofibrate was non-
competitive with respect to the substrate ATP.
Discussion
Metabolic studies suggest that the antilipolytic action
of clofibrate is due to reduced intracellular cAMP
concentration (D'Costa and Angel 1975). The level
of cAMP achieved is the net balance between syn-
thesis via adenylate cyclase and degradation by
cAMP phosphodiesterase. An influence of clofibrate
on the latter enzyme system has been excluded in
broken cell preparations of different species (D'Cos-
ta and Angel 1973, D'Costa and Angel 1975). The
aim of this work was to study directly the effects
of clofibrate on the adenylate cyclase system of hu-
man fat cell ghosts.
Human fat cell adenylate cyclase activity is signi-
ficantly depressed by added clofibrate. Dur results
are consistent with the lipolytic studies suggesting
that the antilipolytic action of clofibrate is due to
inhibition of the adenylate cyclase activity: Identical
concentrations of clofibrate inhibit the activity of
the enzyme system to a sirnilar extent as reported
for the antilipolytic activity of this drug in intact
human adipocytes (D'Costa and Angel 1975).
Referenees
&"ett, A.M: The effeet of chlorophenoxyisobutyrie acid
on the release of free fatty acids from isolated adipose
tissue in vitro. Br.J.Pharmaeol. 26: 363-371 (1966)
Ba"ett, A.M., I.M. Thorp: Studies on the mode of aetions
of clofibrate. Effects on hormone-induced ehanges in
plasma free fatty acids, cholesterol phospholipids and
total esterified fatty acids in rats and dogs. Br.J.Phar-
maeol.Chemother. 32: 381-391 (1968)
303
5 mM 20 mM
1.16 ± 0.19 2.46 ± 0.25
1.35 ± 0.14
(-45%) (-46%)
2.09 ± 0.18 3.07 ± 0.18
1.63 ± 0.24
(-45%) (-47%)
1.74 ± 0.24 2.55 ± 0.26
1.41 ± 0.22
(-45%) (-45%)
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
The rapid onset of clofibrate action suggests that
inhibition of the human fat cell adenyla.te cyclase
system does not depend on transformation of the
drug into an effective derivate. This is in contrast
to the proposals of Greene, Herman and Zakim
(1970). These authors reported an inhibition of the
enzyme system in adipose tissue of clofibrate-fed
rats, but failed to observe an effect on the in vitro
adenylate cyclase activity.
The fact that non-stirnulated, NaF and hormone ac-
tivated enzyme activities are inhibited to the same
extent indicates that clofibrate primarily tends to
affect the catalytic subunit of the enzyme system.
This finding implies that this compound does not
considerably influence the initiation and transduc-
tion of hormonal signals or the mechanisms where-
by fluoride actiaates the enzyme. The inhibition
of clofibrate is non-competitive with respect to sub-
strate (ATP) and cofactors (Mg2 +).
The mechanisms of clofibrate action are, therefore,
similar to those reported for tetracycline (Counis,
Koumanov, Raulin and In!ante 1973), but quite dif-
ferent from the action of other modulators of the
enzyme system such as phenothiazines (Wal!! and
Jones 1970), gramicidine S and cobramine B (Wolf!
and Jones 1971).
In conclusion, our result support the hypothesis that
the antilipolytic action of clofibrate is due to sup-
pression of cyclic AMP production by inhibition of
the catalytic subunit of membrane-bound adenylate
cyclase activity.
D'Costa, M., A. Angel: Inhibition of hormone stimulated
lipolysis by clofibrate: meehanism for its hypolipidemic
effeet. Clin.Res. 21: 884 (1973) (Abstr.)
D'Costa, M., A. Angel: Inhibition of hormone-stimulated
lipolysis by clofibrate. A possible mechanism for its hy-
polipidemic action. J.Clin.lnvest. 55: 138-148 (1975)
Counis, R., K. Koumanov, J. Raulin, R. Infante: Interpreta-
tion du role antilipolytiquc dc la tetracycline inhibition
de I'adcnylatc cyclase in vitro. EULJ.Biochcm. 37: 244-
247 (\973)
304 D.P. Bloxham, J.T.R. Fitzsimons, D.A. York
Fredrickson, D.S.: A physician's guide to hyperlipidemia.
In: Modem concepts of cardiovascular disease. R.C.
Schlant, ed. American Heart Association Inc., New York
41: 31-35 (1972)
Greene, H.L., R.H. Herman, D. Zakim: The effcct of c10-
fibrate on rat tissue adenyl cyclase. Proc.Soc.Exp.Biol.
Med. 134: 1035-1038 (1970)
Havel, R.J., J.P. Kane: Drugs and lipid metabolism. Ann.
Rev.Pharmacol. 13:287-308 (1973)
Lowry, O.H., N.J. Rosebrough, A.L. Farr, R.J. RandalI:
Protein measurement with the Folin phenol reagent.
J.BioI.Chem. 193: 265-275 (1951)
Pohl, S.L., L. Birnbaumer, M. Rodbell: The glucagon-sen-
sitive adenylate cyclase system in plasma membranes
of rat liver. J.BioI.Chem. 211: 20-30 (1970)
Rodbell, M.: Metabolism of iso la ted fat ceHs. I. Effects of
Requests for reprints should be addressed to: Dr. H. Kather, Dr. B. Simon, Klinisches Institut rur Herzinfarktforschung an
der Medizinischen Universitätsklinik Heidelberg, Bergheimerstr. 58, 0-69 Heidelberg, Germany
Lipogenesis in Hepatocytes of Genetically Obese Rats
D,P. Bloxham, J.T,R. Fitzsimons and D.A. York
Department of Physiology & Biochemistry, University of Southampton, Southampton, England
Summary
A simple method for the preparation of hepatocytes in good
yield from obese rats is described, Lipogenesis from [U_14C}-
lactate, [U-14C}-glucose and tritiated water has been inves-
tigated in hepatocytes prepared from both geneticaHy obese
'fatty' rats and their lean Iittermates. Hepatocytes from obese
rats dt:monstrate eIevated rates of fatty acid and sterol syn-
thesis in contrast to hepatocytes from lean animals. The rate
of fatty acid synthesis is sensitive to the dietary status of
the animal prior to preparation. The enhanced Iipogenesis
in hepatocytes from obese rate depends on lactate as a source
of carbon rather than glucose. Th" role of the liver in genetic
obesity is discussed with particular reference to the major
precursor for hepatic Iipgcnesis.
Key-Words: Obese Rats - Hepatocytes - Lipogenesis -
Glucose - Lactate
Introduction
The perfused liver and isolated hepatocyte prepara-
tions have been used extensively in recent years to
investigate aspects of liver metabolism. As a net rate
of gluconeogenesis is predominant in the livers of
both starved and fed rats (Clark, Bloxham, Holland
and Lardy 1974) it is unlikely that glucose can be
a major precursor of the acetyl CoA necessary for
fatty acid synthesis. A number of reports have con-
Received: 16 Apr, 1976 Accepted: 10 Jan. /977
hormones on glucose metabolism and Iipolysis. J.Bio\.
Chem. 239: 375-380 (1964)
Salomon, Y., C. Londos, M. Rodbell: A highly sensitive
adenylate cyc\ase assay. Anal.Biochem. 58: 541-548
(1974 )
Smigura, F.C., J. Farkas, A. Angel: The effect 01' c\ofibrate
on sterol synthesis and storage in various rat tissues.
Clin.Res. 20: 946 (1973) (Abstr.)
Wolf!. J., A.B. Jones: Inhibition of hormone-sensitive ade-
nylate cyc\ase by phenothiazines. Proc.Nat.Academ.Sci.
65: 454459 (1970)
Wolf!. J., A.B. Jones: The purif1cation of bovine thyroid
plasma membranes and the properties of membrane-
bound adenyl cyclase. J.BioI.Chem. 246: 3939-3947
(1971 )
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Horm. Metab. Res. 9 (1977) 304-309
© Georg Thieme Verlag Stuttgart
firmed this observation and suggest that lactate rather
than glucose might be the major precursor of the ace-
tyl CoA (Clark, Rognstad and Katz 1974, Brnnengra-
ber, Boutry and Lowenstein 1973, Salmon, Bowen and
Hems 1974) in the rat and mouse.
The genetically obese 'fatty rat' provides an excellent
experimental model for further investigation of liver
metabolism. These animals are characterised by ele-
vated serum insulin and triglyceride levels even after
starvation (Barry and Bray 1969), by hypersecretion
of very low density lipoprotein (Schonfeld and Pfle-
ger 1971) and by enhanced activities of various lipo-
genie enzymes (Martin 1974, Taketomi, Ishikawa and
Iwatsuka 1975).
The availability of isolated cells provides a convenient
mechanism for studying metabolie processes. Our ini-
tial purpose was to isolate hepatocytes from obese
rats, to demonstrate that these cells possessed an en-
hanced lipogenic capacity in comparison to their lean
littermate eontrols and to examine whether glucose
or lactate was the preferred carbon source vor lipo-
genesis.
Materials and Methods
90-120 day old genetically obese (fa/fa) rats (400·500 gm)
or their lean Iitter mate controls (Fa/" ) (250-300 !!) wnl'
USL'd. ThL' ammaJs \\L'ft' fL'd t'itlll" I"h",,,(,,[\' Li1l)\\ 11',,( ,'''''1,'''1