Regulation of lipolysis and cyclic AMP synthesis through energy supply in isolated human fat cells

385

Biochimica et Biophysics @ Elsevier/North-Holland

Acta, 486 (1977) 385-398 Biomedical Press

BBA 56923

REGULATION OF LIPOLYSIS AND CYCLIC AMP SYNTHESIS ENERGY SUPPLY IN ISOLATED HUMAN FAT CELLS

YVES GIUDICELLI, RENE and ROGER NORDMANN

PECQUERY,

DANIELE

PROVIN,

BRIGITTE

THROUGH

AGLI

Service de Biochimie de la Faculte’ de Me’decine de Paris-Ouest and Groupe de Recherches de I’INSERM sur le Mktabolisme Interme’diare, Laboratoire de Biochimie du Centre Hospitalier, 78303 Poissy (France) (Received

July 5th, 1976)

Summary The effects of glucose and of various inhibitors of glycolysis or of oxidative phosphorylation on stimulated lipolysis and on intracellular cyclic AMP and ATP levels were investigated in isolated human fat cells. The glycolysis inhibitors, NaF and monoiodoacetate, inhibited epinephrineor theophylline-stimulated lipolysis and parallely reduced the intracellular cyclic AMP and ATP levels; however, neither NaF nor monoiodoacetate significantly affected dibutyryl cyclic AMP-induced lipolysis. Removal of glucose from the medium also reduced the rate of epinephrine-stimulated lipolysis and the intracellular cyclic AMP and ATP levels but failed to modify the lipolytic activity of dibutyryl cyclic AMP. The oxidative phosphorylation inhibitors, antimycin A and, under fixed conditions, 2,4dinitrophenol also strongly decreased the adipocyte cyclic AMP and ATP levels but inhibited as well the rate of epinephrine- and of dibutyryl cyclic AMP-induced lipolysis. N-Ethylmaleimide, a mixed glycolysis and oxidative phosphorylation inhibitor, not only reduced the intracellular cyclic AMP and ATP levels and epinephrine- or theophylline-induced lipolysis, but also that stimulated by dibutyryl cyclic AMP. When glycolysis was almost fully inhibited, human fat cells were insensitive to epinephrine but remained fully responsive to dibutyryl cyclic AMP. These results, showing a relationship between ATP availability, cyclic AMP synthesis and lipolysis, suggest a different ATP requirement for cyclic AMP synthesis and triacylglycerol lipase activation, a difference which could explain why ATP issued from glucose breakdown appears to be a determinant factor for cyclic AMP synthesis, but not for triacylglycerol lipase activation in human fat cells.

386

Introduction The antilipolytic effects exerted by different inhibitors of glycolysis and oxidative phosphorylation on rat adipose tissue have been widely studied by Fassina et al. [l--5]. Although the effects of these inhibitors on intratissue ATP levels were not investigated, these authors recently suggested that adenylate cyclase activity and triacylglycerol lipase activation could be differently dependent on ATP provided either by glycolysis or by oxidative phosphorylation [ 51. In human adipose tissue, glucose metabolism [6,7] and sensitivity to lipolytic hormones [8--111 are markedly different from those described in the rat. Because of these differences, and in order to determine to what extent the lipolytic process in human fat cells is energy-dependent, we investigated the influence of glycolysis or oxidative phosphorylation inhibitors on the lipolytic activity of isolated human fat cells. In a preliminary communication [ 121, we reported, as Fassina et al. [ 3,4] showed in the rat, that the glycolysis inhibitors, NaF and monoiodoacetate, and the oxidative phosphorylation inhibitor antimycin A severely depressed epinephrine- or theophylline-induced lipolysis. However, in opposition to the findings of Fassina et al. [2,3] concerning rat adipose tissue, we were unable to observe a significant inhibition of dibutyryl cyclic AMP-induced lipolysis with NaF and monoiodoacetate; moreover, lipolysis induced by epinephrine, theophylline or dibutyryl cyclic AMP was either unaffected by rotenone or slightly reduced by 2,4_dinitrophenol. These results led us to investigate whether the antilipolytic effects shown by some of these inhibitors could be linked to their interference with energy availability and to determine whether the energy issued from glucose metabolism may assume, in human fat cells, a special role in the regulation of the main steps of the lipolytic process, i.e. cyclic AMP synthesis and triacylglycerol lipase activation. This was accomplished by studying the influence of glucose and of the different above mentioned metabolic inhibitors on the lipolytic activity as well as on the intracellular cyclic AMP and ATP levels in human fat cells exposed to lipolytic agents acting before or beyond the synthesis of cyclic AMP. Adenylate cyclase stimulation was induced by epinephrine [ 13,141, phosphodiesterase inhibition by theophylline [ 153 and triacylglycerol lipase activation by N6,2,0dibutyryl cyclic AMP, which mimicks the effects of endogenous cyclic AMP

[161. Material

and Methods

Material Collagenase was obtained from Worthington Biochemical Corporation, bovine serum albumin (Fraction V, fatty acid poor), L-epinephrine bitartrate, N6,2,0dibutyryl cyclic AMP (monosodium salt), antimycin A and 2,4-dinitrophenol from Calbiochem, theophylline, monoiodoacetate, NaF and N-ethylmaleimide from Merck. Enzymes, coenzymes and substrates were from Boehringer, Mannheim. Cyclic AMP, 3H-labeled cyclic AMP ammonium salt (speci-

fit activity 25 Ci/mmol) and cyclic AMP binding protein (bovine muscle) were purchased from the Radiochemical Centre, Amersham.

skeletal

Preparation and incubation of human fat cells Human omental adipose tissue was obtained from 25 adult subjects having no clinical evidence of endocrine disease and undergoing abdominal surgery. Patients fasted overnight were premeditated before the operation with atropine and pethidin. The induction of anesthesia as well as the methods used for collection and transport of adipose tissue have been described elsewhere [ 171. Isolated fat cells were prepared according to Rodbell [18] with minor modifications [ 121. Aliquots (0.2 ml) of the cell suspension equivalent to 100 + 20 mg cell lipid were transferred to polyethylene incubation flasks containing, unless otherwise stated, Krebs-Ringer bicarbonate buffer (pH 7.4) [ 191, albumin (4%) and glucose (5 mM), the final volume being 5 ml. In some experiments, however, a glucose-free medium or 1% albumin buffer solution was used. In experiments in which glucose uptake, lactate and pyruvate output were studied, streptomycin (0.2 mg/ml) was added to the incubation medium. Incubations were carried out in a shaking water bath (100-120 cycles/mm) for 3 h at 37°C under 95% 02--5% COZ. Unless otherwise stated, all the reagents tested were added at the start of the final incubation in saline or ethanol, to the final concentrations indicated. The same volumes of saline or ethanol were added to the control samples. Under these conditions, addition of ethanol failed to affect the lipolytic response of the fat cells. When intracellular levels of cyclic AMP were determined, fat cells were first incubated in the presence or absence of the different inhibitors. After a l-h preincubation, epinephrine (50 PM) and theophylline (5 mM) were added and the assays further incubated. Intracellular cyclic AMP levels were determined 30 min later. This time was found to be optimal under the present experimental conditions, confirming thus the previous data of Burns et al. [ 10,201 according which the maximal rise in cyclic AMP levels occurs in human fat cells within 30 min following the addition of the stimulating compounds. Analytical procedures At the end of the incubation periods, cells and medium were immediately separated by filtration. The rate of lipolysis, which was linear during the 3-h period of incubation (results not shown) was determined by measuring the amount of glycerol and free fatty acid released into the medium. Glucose uptake was estimated by measuring glucose disappearance from the medium. Glycerol [21] and glucose [22] were assayed enzymatically and free fatty acids were titered according to the method of Dole and Meinertz [ 231. Results are expressed as pmol of glycerol or of free fatty acid released or as pmol of glucose taken up per g cellular lipid during 3 h of incubation. Lactate and pyruvate released in the medium were determined enzymatically as described previously [24] and results are expressed as pmol produced per g cellular lipid during 3 h of incubation. When intracellular ATP or cyclic AMP levels were determined, the content of each flask was rapidly centrifuged at 0-4°C and the medium removed by

388

aspiration. The remaining cells were homogenized either in 0.6 M ice-cold perchloric acid and centrifuged (assay of ATP) or in ice-cold 50 mM Tris * HCl buffer pH 7.5 containing 4 mM EDTA, boiled for 3 min, cooled and centrifuged (assay of cyclic AMP). ATP was determined enzymatically in the intermediary deproteinized phase, according to a modification [25] of the method of Denton et al. [26] and results expressed as nmol ATP per g cellular lipid. Determination of cyclic AMP was performed using the protein kinase radio competitive binding assay of Gilman [27] with the following minor modifications. Supernatants were evaporated to dryness and redissolved in 50 mM Tris HCl, 4 mM EDTA buffer, pH 7.5. The assay system consisted in 4.5 pg bovine skeletal muscle binding protein, 0.9 pmol 3H-labeled cyclic AMP both in 50 mM Tris - HCl, 4 mM EDTA buffer, pH 7.5 and 50 ~1 of unlabelled cyclic AMP (standards and assays), the final volume being 200 ~1. Reaction was started by the addition of the binding protein and incubated 120 min at 0°C. 100 i.rl charcoal suspension were added to each tube which was then mixed. After centrifugation, supernatants were removed and radioactivity determined in a Packard Tricarb liquid scintillation spectrometer, using 0.4% Permablend III, 20% ethanol and 7% methanol in toluene as scintillation solution. Overall recovery of synthetic cyclic AMP added to control samples was between 90 and 95%. The results for this recovery, expressed as pmol cyclic AMP per g cellular lipid, were corrected in each experiment. The amount of cellular lipids present in each flask was determined gravimetrically. Cells were twice extracted with chloroform and a portion of the extract was evaporated to dryness and weighed. Results are given as mean values If: S.E. and Student’s t-test was used for comparison of mean values. Results The results in Table I show, in accordance with our previous findings [ 121: (a) the inhibition by the glyeolysis inhibitors (NaF and monoiodoacetate) of epinephrineand theophylline(but not of dibutyryl cyclic AMP) induced lipolysis; (b) the inhibition of the lipolytic action of epinephrine, theophyline and dibutyryl cyclic AMP by both N-ethylmaleimide and antimycin A; (c) the failure of 2,4dinitrophenol to affect epinephrine-induced lipolysis and the slight ~tagonistic effect of this compound on the lipolytic action of theophylline and dibutyryl cyclic AMP. To assess the mechanism of the antilipolytic effect induced by some of the inhibitors tested, we studied the modifications induced by these inhibitors on the intracellular accumulation of cyclic AMP in response to the simultaneous addition of epinephrine (50 PM) and theophylline (5 mM) (Tables II and III). In order to determine whether this antilipolytic effect could be related to the inhibition of glycolysis or of oxidative phosphorylation, we also investigated, in the presence of epinephrine (50 PM) or dibutyryl cyclic AMP (1 mM), the modifications induced by these inhibitors on some metabolic intermediates reflecting the activity of both pathways (Tables II and III). As shown in Table II, addition of epinephrine and theophylline induced a

I RATE

OF EPINEPHRINE

(50 PM), TIiEOPHYLLINE

(5 mM) OR

22.55 f 3.03.

Dibutyryl ~yclle AMP

* P < 0.001 ** 0.02

om

20.42 f 1.92

Theophylline

-_~______

0

16.82 ” 3.29 *

9.52 k 0.67 *

3.61 i 0.40 *

--.--.-

NaF

Additian to the medium:

.-^--

17.25 L 2.63 -F

12.25 !: 1.33 *

8.25 I: 0.91 *

Monaiodoacetate l~...“l”“.-._-

Glycerol release (PM/~ cell lipid per 3h) ,-” -~

14.70 f 1.51

~

Epinephrine

--

tions.

-~

4.99 :k 0.67 *

3.59 f 0.65 *

6.53 * 0.48 *

N-ethyl maleimide .._^-^..“-- -- __~-

4.91 f 0.39 *

7.25 +- 1.12 *

4.01 + 0.55 *

Antimycin A

_~_.____

16.21 f 2.34 **

16.40 t 1.35 **

12.06 I 1.55 ***

- ---_-

2,4-Dinitrophenol ^-____-

_.~___

Fat cells were incubated in Krebs-Ringer bicarbonate buffer containing glucose (5 mMf, albumin (4%, wiv) and, when indicated, epinspbrlne (50 PM), tbeophylline (5 mM), dibutyryl cyclic AMP (1 mMf and the metabolic inhibitors at the following concentrations: NaF, 10 mM; monoiodoacetate, 1 mM; iV-ethylmaleimide, 0.45 mM; antimycin, 20 HM; 2.4-dtit~ophe~ol. 1 mM. The determination of glycerol was performed 3 h later. Each vahae represents the mean + S.E. of IO determina-

EFFECTS OF GLycoLYs~s AND OXIDATIVE PHOSPHORYLATION INIIIBITORS ONTHE DIBUTYRYLCYCLIC AMP (1 mM) STIMULATED LII’QSYSlS IN HUMAN FAT CELLS

TABLE

ADIPOCYTES

ON LIPOLYSIS.

HUMAN

INHIBITORS INCUBATED

CYCLIC OF EPINEPHRINE

AMP AND ATP LEVELS,

IN THE PRESENCE

INTRACELLULAR

GLUCOSE

UPTAKE,

PYRUVATE

(50 PM) OR DIBUTYRYLCYCLIC

AND AMP

c 1.12

15.73 +- 3.57 ***

17.31 i 2.68 **’

Dibutyryl cyclic AMP C monoiodoacetate (15)

63.42

62.87

77.82

t 5.90 ***

+_7.60 ***

+ 8.86

8.44 i 2.15 *

r 1.64 *

8.15 i 1.21 *

15.71

* P < 0.001 vs. epinephrine ** P < 0.001 vs.dibutyryl cyclic AMP. *** P > 0.05 vs. dibutyryl cyclic AMP.

20.81 * 2.17

5.03 i 0.36 *

Dibutyryl cyclic AMP + NaF (15)

5.94 f 0.32 *

Epinephrine + N-ethylmaieimide (10)

Dibutyryl cyclic AMP (15)

3.45 * 0.59 *

Epinephrine + monoiodoacetate (10)

29.10

+ 1.46

11.44

---

3b)

3b)

3.17 f 0.50

(&M/g cell lipid/

&M/g cell lipid/

1.93 2 0.18

Free fatty acid release

Glycexol l%lWASe

Epinephrine + NaF (10)

Epinephrine (10)

0 (5)

Addition to the medium

N.D.

N.D.

N.D.

235 ? 32 *

261 ? 66 +

217 + 44 *

576 t 76

81 +_16

(PM/g cell lipid)

Cyclic AMP level

5*

6’

7

305

272

4’s

7*”

96 + 10

4126*

47+

37i.

942

225 + 28

+ 3.13 *

I 2.97

--

+ 7.08

1.13 i 0.87 **

4.81 + 1.90 **

23.01

N,D.

7.68 + 2.19 *

12.01

30.88

20.19

+ 3.66

&M/g cell Lipid/

(nM/g cell lipid) 3h) -.--____

Ghxose uptake

ATP level

*

0.22 ?r 0.08 *’

0.59 + 0.10 *

1.29 1. 0.11

N.D.

2.00 _+0.72

1.42 I 0.78 *

3.82 i 0.49

2.30 i 0.51

‘&M/g cell lipidj3h)

Pyruvate output

? 2.39

?; 2.13

?1 2.33

2.50 _t 0.67 **

3.29 tl 0.92 **

16.32

N.D.

1.32 ?: 0.29 *

3.19 i- 0.90 *

20.41

13.50

lipid/3h) .- ---

f&M/g cell

Lactate output

+

pyruvate

t 2.41

f. 2.05

+ 2.54

2.72 + 0.93 **

3.88 + 1.14 **

17.61

N.D.

3.32 + 0.52 *

4.61 i- 1.14 *

24.23

15.80

3W

(&M/g cell lipid/

output

La&ate

Experimental conditions were as in Table I except for the determination of cyclic AMP which was determined as followed: fat cells were first incubated in KrebsRinger bicarbonate buffer containing glucose (5 mM), albumin (4%. w/v) and, where indicated, the metabolic inhibitors at the same concentrations as in Table I. After a preincubation of 1 h, epinephrine (50 FM) and theophylline (5 mM) were added and the assays further incubated; determination of cyclic AMP was performed 30 min later. Each value represents the mean i S.E. with the number of determinations in parentheses. N.D.: not determined.

(1 mM)

OUTPUT

BY ISOLATED

OF GLYCOLYSIS

EFFECTS

LACTATE

TABLE II

391

7.1-fold increase in the intracellular cyclic AMP level. Confirming the previous reports of Carlson et al. [ 28,291, this reponse of human fat cells contrasts with the much more striking increase of cyclic AMP usually found in rat fat cells [30]. Although the experimental conditions for the determination of cyclic AMP and lipolysis were different regarding both the incubation times and the lipolytic agents used (see Material and Methods), it is of interest to note that NaF, monoiodoacetate and N-ethylmaleimide induced parallel changes in the intracellular cyclic AMP and ATP levels as well as in the magnitude of epinephrine-stimulated lipolysis. Under these conditions, these effects were accompanied by a strong reduction of glucose uptake, pyruvate and lactate output, thus confirming the occurrence of glycolysis inhibition (Table II). These latter metabolic disturbances were also induced by NaF and monoiodoacetate when fat cells were incubated with dibutyryl cyclic AMP in the place of epinephrine (Table II); however, no significant inhibition of the lipolytic activity of dibutyryl cyclic AMP was observed under these conditions. As shown in Table III, the oxidative phosphorylation inhibitor antimycin A severely impaired the intracellular cyclic AMP and ATP levels as well as the adipocytes extramitochondrial redox state reflected by the lactatelpyruvate ratio. In fact, cyclic AMP accumulation induced by epinephrine and theophylline was strongly reduced (67%); furthermore, the marked inhibitory effect induced by antimycin A on epinephrine-induced lipolysis was accompanied by a 50% decrease in the intracellular ATP level and a 17-fold increase in the lactate/ pyruvate ratio. Under the same experimental conditions, 2,4dinitrophenol which failed to effect epinephrine-stimulated lipolysis (Table I) had no significant effect on the intracellular cyclic AMP and ATP levels nor on the lactate/ pyruvate ratio (Table IV). However, when the albumin concentration in the medium was lowered from 4 to l%, 2,4dinitrophenol induced a marked uncoupling effect which was accompanied by both a strong inhibition (61%) of epinephrine-stimulated lipolysis and a parallel decrease (58%) in the cyclic AMP accumulation in response to epinephrine and theophylline (Table IV). Since the above data give no indication on the relative significance of glycolysis and oxidative phosphorylation in the regulation of epinephrine-induced lipolysis, we investigated the influence of glucose on both the ATP level and the lipolytic activity of adipocytes studied either in the basal state or in the presence of epinephrine. As shown in Table V, the basal ATP level in adipocytes incubated in a glucose free medium was markedly reduced compared with the basal ATP level found in adipocytes incubated with glucose; furthermore, in the absence of glucose, ATP was not significantly affected by epinephrine, which induced, on the contrary, a 50% decrease in the ATP level of adipocytes incubated with glucose. Glucose also influenced the rate of lipolysis, stimulation by epinephrine being 3.5-fold in the absence of glucose and 5.6-fold in the presence of glucose. Changes in the intracellular cyclic AMP level and in the glycerol release induced by the simultaneous addition of epinephrine and theophylline were similarly influenced by glucose, the omission of glucose resulting in a 50-60% decrease in both the lipolytic activity and cyclic AMP level (Table VI). The following experiments were carried out to determine whether inhibition of oxidative phosphorylation may further increase the antagonistic effect

392

393 TABLE

IV

EFFECTS

OF 2,4-DINITROPHENOL

LAR CYCLIC ADIPOCYTES

ON EPINEPHRINE-STIMULATED

LIPOLYSIS,

AMP AND ATP LEVELS AND LACTATEIPYRUVATE INFLUENCE OF ALBUMIN CONCENTRATION

RATIO

Experimental conditions as in Table II. except for the metabolic inhibitor (1 mM). Each value represents the mean f S.E. of 5-15 determinations. Addition to the medium

Albumin

4% Epinephrine + 2,4-dinitrophenol

Epinephrine

Glycerol release WM/g cell lipid/3h)

11.98

Cyclic AMP level (pM/g cell lipid)

572

ATP level (nM/g cell lipid)

90

Lactatelpyruvate ratio

TABLE

6.2

k

which

Albumin

1.71

11.52

HUMAN

was. 2,4dinitrophenol

1%

Epinephrine

9.06 * 1.55 (P > 0.05)

INTRACELLU-

IN ISOLATED

+

Epinephrine + 2,4-dinitrophenol 1.45

4.49 + 0.36 (P < 0.001)

* 76

408 ? 87 (P > 0.05)

356

* 34

148 k 12 (P< 0.001)

f

8

81 ? 9 (P > 0.05)

97

+ 10

18r 3 (P < 0.001)

?

2.8

7.8 t 1.9 (P > 0.05)

+

50.1 t 9.9 (P < 0.001)

6.5

1.7

V

INFLUENCE OF GLUCOSE ON THE INTRACELLULAR DUCED BY EPINEPHRINE (50 PM) IN ISOLATED HUMAN

ATP LEVEL FAT CELLS

AND

ON

LIPOLYSIS

IN-

Fat cells were incubated for 3 h in Krebs-Ringer bicarbonate buffer containing albumin (4%. w/v) and, when indicated glucose (5 mM) and epinephrine (50 PM). At the end of the incubation, the intracellular ATP level and the glycerol released into the medium were determined. Each value represents the mean + S.E. of 5 determinations. Addition to the medium

None

-

None ATP level (nM/g cell lipid)

92

Glycerol release (PM/g cell lipid/3h)

TABLE

f 18

1.48 *

0.28

Glucose

Epinephrine

None

67 + 11 (P > 0.05)

211

5.26 r 0.92 (P < 0.001)

Epinep brine * 16

2.47 *

108 k 17 (P < 0.001)

0.35

13.83 f 1.20 (P < 0.001)

VI

INFLUENCE OF GLUCOSE ON LIPOLYSIS AND ON CYCLIC AMP ACCUMULATION INDUCED EPINEPHRINE (50 PM) AND THEOPHYLLINE (5 mM) IN ISOLATED HUMAN FAT CELLS

BY

Fat cells were incubated in Krebs-Ringer bicarbonate buffer containing albumin (4%. w/v) and, when indicated, glucose (5 mM). After a preincubation of 1 h, the glycerol released into the medium was determined. Epinephrine (50 PM) and theophylline (5 mM) were then added and the assays further incubated. Determination of intracellular cyclic AMP levels and glycerol in the medium was performed 30 min later. Each value represents the mean + S.E. of 10 determinations. Addition to the medium

None

Glucose Epinephrine theophylline

None

Glycerol release (PM/g ceil lipid/30 Cyclic AMP @M/g cell lipid)

0.50

*

0.08

min) 84

* 10

1.70 ? 0.23 (P < 0.001) 256 + 51 (P < 0.001)

+

Epinephrine theophylline

None

0.53 107

f

0.15

? 17

3.46 * 0.31 (P < 0.001) 636 f 53 (P < 0.001)

+

394 TABLE VII INFLUENCE OF ANTIMYCIN A ON THE EFFECTS EPINEPHRINE (50 J&T) OR DIBUTYRYLCYCLIC ISOLATED HUMAN FAT CELLS

INDUCED BY GLYCOLYSIS AMP (1 mM) STIMULATED

INHIBITION LIPOLYSIS

ON IN

Fat cells were incubated for 3 h in Krebs-Ringer bicarbonate buffer containing albumin (4%. w/v) and when indicated glucose (5 mM), epinephrine (50 MM), dibutyryl cyclic AMP (1 mM), NaF (10 mM) and antimycin A (20 PM). Each value represents the mean f S.E. of five determinations. Expt.

-_.-__

Addition to the medium

Glycerol release (PM/~ cell lipid per 3h)

____~

1

NaF NaF + antimycin A Epinephrine Epinephrine + NaF Epinephrine + NaF + antimycin A Dibutyryl cyclic AMP Dibutyryl cyclic AMP + NaF Dibutyryl cyclic AMP + NaF + antimycin A

2 3 4 5 6 7 8 a b c d e f

P < 0.001 P < 0.001

P < P < P < P <

vs. Expt. 1 and 3. vs. Expt. 3 and 4: non significant vs. 0.001 vs.Expt. 1; non significant vs. Expt. 0.001 vs. Expt. 1.6 and 7. 0.001 vs. Expt. 3, non significant vs. Expt. 0.001 vs. Expt. 3; non significant vs. Expt.

Glucose (5 mM) _ -_--.__~~~~_.

No &xose

1.94 1.66 12.87 6.16 2.11 22.31 18.03 4.24

1.22 0.95 5.60 1.69 1.29 17.81 17.77 3.30

?: 0.33 IO.27 Il.34 i 1.50 f- 1.11 + 2.89 C 3.57 +_0.86

a b ’ d

_ ..-- _....

f 0.35 i: 0.30 i 0.83 L 0.31 ’ 10.24 f F 1.23 i 0.85’ i 0.47 d

Expt. 1. 6. 1. 1 and 4.

induced by glycolysis inhibition on epinephrine-stimulated lipolysis (Table VII). Since, as shown in Table II, glucose uptake and glycolysis were not completely abolished by NaF, these experiments were performed in both the presence and absence of exogenous glucose. When NaF and glucose were both present, addition of antimycin A did not modify the basal glycerol release. Under these conditions, addition of epinephrine induced a 3.2-fold increase of lipolysis, an effect which was almost completely prevented by antimycin A. On the contrary, when fat cells were incubated in a glucose free medium but in the presence of NaF, addition of epinephrine or epinephrine plus antimycin A had no more significant effect on lipolysis. Under these conditions, the strong inhibitory effect of antimycin A on dibutyryl cyclic AMP-induced lipolysis remained unchanged. Discussion The regulation of the lipolytic process in human adipose tissue is a subject of dual experimental interest. Firstly, the major factor regulating the tissular fatty acid utilization, the free fatty acid blood level, is controlled by the rate of fatty acid release from fat cells; disorder8 in adipose tissue lipolytic activity may thus play an impo~ant role in the pathogenesis of metabolic diseases such as atheroma, obesity, diabetes and ketosis. Secondly, the effects of hormones or drugs, which provide a valuable tool for the study of the mechanisms involved in the lipolytic process, vary greatly from human to animals: for example, ACTH, glucagon or growth hormone which are potent lipolytic hormones in rat fat cells, are ineffective in human adipocytes [9,10]. Membrane receptors sensitive

395

to a-adrenergic agents which inhibit lipolysis are present in human adipocytes, but are lacking in rat fat cells [lO,ll]. Furthermore, NaF, monoiodoacetate, 2,4dinitrophenol and antimycin A, in the same concentrations as those used presently were found to be equally potent inhibitors of norepinephrine, theophylline or dibutyryl cyclic AMP-stimulated lipolysis in rat adipose tissue [2-51, whereas, under similar experimental conditions, the present results show that in human fat cells, the antilipolytic effects of these inhibitors are different in magnitude and depend on the lipolytic agent used. Despite these species differences, Khoo et al. [31] have recently demonstrated that the lipolytic process, in human adipose tissue, follows the same metabolic pathways as those occurring in rat fat cells, i.e. synthesis of cyclic AMP and activation of triacylglycerol lipase by a cyclic AMP-dependent protein kinase. Accordingly, ATP appears necessary not only for cyclic AMP synthesis but most probably also for the activation of triacylglycerol lipase and, therefore, stimulation of lipolysis in human fat cells should require, as in the rat [l-5], a continuous supply of energy. Although the inhibitors used here might have other actions influencing lipolysis than those on ATP synthesis, it appears from the present data that alteration of energy equilibrium in human fat cells interferes indeed with the lipolytic process at two different levels, i.e. both before and beyond cyclic AMP synthesis. It suggests, furthermore, that these different levels may be differently dependent on the metabolic pathways providing energy to human fat cells. In fact, the present data showing that the glycolysis inhibitors NaF, monoiodoacetate and N-ethylmaleimide reduce to a similar extent the rate of epinephrine-stimulated lipolysis and the intracellular levels of both cyclic AMP and ATP, strongly suggest that these inhibitors act on epinephrine-stimulated lipolysis through their interference with cyclic AMP synthesis. These effects, which contrast with the well-known stimulating action of NaF on adenylate cyclase in broken cell systems incubated in the presence of a continuous ATP generating system [ 14,321, have been already reported in intact rat fat cells or tissue [3,33] and appear, according to the present results, to be related to the fall in intracellular ATP level. This reduction of ATP level could be the consequence of the uncoupling action exerted by increased intracellular accumulation of free fatty acids [25] resulting from the association of increased lipolysis due to epinephrine and decreased reesterification induced by glycolysis inhibition. However, Table V shows that, in the absence of lipolytic agents, the omission of glucose also reduces the basal intracellular ATP level; this suggests that this fall in ATP is more likely to be related to decreased energy availability caused by glycolysis inhibition, than to uncoupling induced by intracellular free fatty acid accumulation. This is furthermore supported by the fact that exogenous glucose induces similar changes in the intracellular ATP level, in the rate of epinephrineor of epinephrine plus theophylline-stimulated lipolysis and in the cyclic AMP accumulation in response to epinephrine plus theophylline (Tables V and VI). These data thus emphasize the importance of ATP supplied by glucose oxidation in the regulation of cyclic AMP synthesis in human fat cells. The present results also show that dibutyryl cyclic AMP-induced lipolysis is unaffected by NaF, monoiodoacetate and by the removal of glucose, but markedly inhibited by N-ethylmaleimide. Since the latter compound has been

396

shown to interfere with some mitochondrial dehydrogenases and with the mitochondrial oxidative phosphorylation and translocation process [ 34,351, inhibition of dibuty~l cyclic UP-induced lipolysis by ~-ethylm~eimide cannot be considered solely as the result of glycolysis inhibition. On the contrary, the lack of inhibitory effect of NaF, monoiodoacetate or glucose omission on dibutyryl cyclic AMP-induced lipolysis suggests that ATP issued from glucose oxidation is not essential for the triacylglycerol lipase activation step. Experiments described in Table IV indicate that, in the presence of glucose, the inhibitors of oxidative phosphorylation tested also interfere with the rate of both cyclic AMP synthesis and triacylglycerol hydrolysis in human fat cells. In fact, it was found that, when studied under conditions inducing uncoupling effects (low albumin concentration), 2,4-dinitrophenol was also a potent inhibitor of both epinephrine-stimulated lipolysis and cyclic AMP synthesis, effects which simultaneously disappeared altogether by raising the albumin concentration. These data, which seem related to the ability of albumin to tightly bind nitrophenols [36], and consequently to prevent the penetration of 2,4-dinitrophenol into fat cells, suggest a relationship between uncoupling and inhibition of both cyclic AMP synthesis and lipolysis. This is further supported by the results obtained with antimycin A, which also induces a marked inhibition of epinephrine, theophylline and dibutyryl cyclic ASP-stimulated lipolysis, as well as a strong decrease in both cyclic AMP and ATP intracellular levels. These findings thus provide evidence that, when glucose breakdown is not inhibited, ATP supplied by oxidative phosphorylation plays an important role in the regulation of both the synthesis of cyclic AMP and the activation of triacylglycerol lipase in human fat cells. In contrast, when glucose oxidation is either reduced or almost fully inhibited (Table VII), human adipocytes are completely insensitive to the lipolytic action of epinephrine, but still remain fully responsive to dibutyryl cyclic AMP. Under these conditions, furthermore, the inhibition of oxidative phosphorylation results in a marked reduction of the lipolytic effects of dibutyryl cyclic AMP. Thus it seems that, under severely reduced glucose availability, triacylglycerol lipase activation and consequently triacylgly~erol hydrolysis, is still possible, the ATP required for this process being most probably supplied in sufficient amounts by the mitochondrial oxidation of non-carbohydrate substrates, i.e. mainly fatty acids. Since, as indicated above, energy supplied by glucose breakdown appears to regulate the lipolytic process essentially at the level of adenylate cyclase, the present results suggest that the ATP requirement or concentration dependence is different for cyclic AMP synthesis and triacylglycerol lipase activation and that larger amounts of ATP are probably required for the former rather than for the latter step of the lipolytic process. This would provide additional support for the hypothesis previously formulated by Kuo and Greengard [ 371 and by Fain et al. [ 381, according to which an important part of cyclic AMP issued from adenylate cyclase activation may be inactive either by virtue of comp~timentation or by binding to fat cell proteins. In conclusion, the present report shows that, in human fat cells, the lipolytic process is, as in the rat, energy-dependent and that ATP produced by glucose breakdown is a determinant factor in cyclic AMP sysnthesis, whereas it does not seem to play an important part in the activation of triacylglycerol lipase.

391

It suggests furthermore, that the ATP required for triacylglycerol lipase activation may be sufficiently supplied by the oxidation of non-carbohydrate substrates. This led us to considere the possibility that, besides its essential role in the regulation of lipogenesis [ 391, glucose may be also, at least in vitro, an important factor for the control of catecholamine-induced lipolysis in human fat cells. Acknowledgements We are particularly indebted to Dr. R. Ronat, from the general surgery division and to Dr. D. Lewin, from the department of obstetrics-gynecology of the Centre Hospitalier de Poissy, for their courtesy in making available human adipose tissue, and to Dr. A. Fingerhut for his meaningful contribution to the English version of this study. This work was supported by grants from the Universite Renk Descartes, Paris. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

Fassina, G., Maragno, I. dnd Dorigo, P. (1967) Biochem. Pharmacol. 16,1439-1446 Fassina, G., Dorigo. P., Badetti, R. and Visco, L. (1972) Biochem. Pharmacol., 21.1633-1639 Fassina. G., Dorigo. P.. Perini, G. and Toth, E. (1972) Biochem. Pharmacol., 21. 2295-2301 Dorigo, P., Visco, L., Fiandini. G. and Fassina, G. (1973) Biochem. Pharmacol. 22. 1957-1964 Fassina, G.. Dorigo. P. and Gaion. R.M. (1974) Pharmacol. Res. Commun. 6.1-21 Goldrick, R.B. (1967) J. Lipid Res. 8. 581-588 Patel. MS., Owen. O.E., Goldman, L.I. and Hanson, R.W. (1975) Metabolism 24, 161-173 Gries, F.A. and Steinke, J. (1967) J. CIin. Invest. 46,1413-1421 EffendiC. S.. Alm, B. and Low. H. (1970) Harm. Metab. Res. 2.287-291 Bums, T.W., Langley, P.E. and Robison, G.A. (1971) Ann. N.Y. Acad. Sci. 185.115-128 Burns, T.W., Langley, P.E. and Robison, G.A. (1972) Advances in Cyclic Nucleotide Research, Vol. 1, pp. 63-85, Raven Press, New York Giudiceili. Y., Provin. D. and Nordmann. R. (1975) Biochimie 57, 979-981 Rodbell, M. (1967) J. Biol. Chem. 242.5744-5750 Birnbaumer, L.. PohI. S.L. and RodbeU. M. (1969) J. Biol. Chem. 244,3468-3476 Butcher, R.W. and Sutherland, E.W. (1962) J. Biol. Chem. 237.1244-1250 Blecher, M. (1971) Metabolism 20. 63-77 GiudiceIli, Y.. Provhr. D. and Nordmann, R. (1975) Biochem. Pharmacol. 24.1029-1033 Rodbell. M. (1964) J. Biol. Chem. 239.375-380 Umbreit. W.W.. Burris, R.H. and Stauffer. J.F. (1964) Manometric Techniques, pp. 132. Burgess, Minneapolis Burns, T.W.. Langley, P.E. and Robison, G.A. (1975) Metabolism 24, 265-276 Eggstein, M. (1966) Kiin. Wochenschr. 44, 267-273 Bergmeyer, H.U. and Bemt, E. (1963) in Methods of Enzymatic Analysis, Vol. 1, pp. 123. Verlag Chemie and Academic Press. New York Dole, V.P. and Meinertz, H. (1960) J. Biol. Chem. 235, 2595-2599 Giudicelli, Y., Nordmann, R. and Nordmann, J. (1972) Biochim. Biophys. Acta 280, 393407 Angel, A., Desai, K.S. and Halperin. M.L. (1971) J. Lipid Res. 12, 203-213 Denton, R.M., Yorke, R.E. and Randle, P.J. (1966) Biochem. J. 100,407-419 Giiman. A.G. (1970) Proc. Natl. Acad. Sci. U.S. 67. 305-312 Carlson. L.A., Butcher, R.W. and MicheIi, H. (1970) Acta Med. Stand. 187. 525-528 Carlson, L.A. and Butcher, R.W. (1972) Advances in Cyclic Nucleotide Research, Vol. 1. pp. 87-90, Raven Press, New York Butcher, R.W., Baird, C.E. and Sutherland, E.W. (1968) J. Biol. Chem. 243,1705-1712 Khoo, J.C., Aquino, A.A. and Steinberg, D. (1974) J. Clin. Invest. 53, 1124-1131 Vaughan, M. and Murad, F. (1969) Biochemistry 8, 3092-3099 Kuo. J.F. and De Renzo. E.C. (1969) J. Biol. Chem. 244. 2252-2260 McGivan, J.D., Grebe, K. and Khngenberg. M. (1971) Biochem. Biophys. Res. Commun. 45, 15331541

398 35 Deb&, R. and Durand, R. (1974) Biochimie 56, 161-170 36 Haurowtiz, F. (1963) in The Chemistry and Functions of Proteins, pp. 239-241, New York 37 Kuo. J.F. and Greengard, P. (1970) J. Biol. Chem. 245, 4067-4073 38 Fain, J.N.. Pointer, R.H. and Ward, W.F. (1972) J. Biol. Chem., 247. 6866+X%72 39 Flatt, J.P. (1970) in Adipose Tissue (Jeanrenaud, B. and Hepp, D. eds.). PP. 93-101, New York

Academic

Press,

Academic

Press,