0021-972x/92/7501-0015$03.00/0 .Journal of Climcal Endocrmology and Metabolism CopyrIght 0 1992 by The Endocrme Society

Mechanisms Subcutaneous JOHAN PETER

HELLMRR, ARNER

Vol. 75, No 1 Printed in U.S A.

for Differences and Omental CLAUDE

MARCUS,

TOMAS

in Lipolysis Fat Cells* SONNENFELD,

between

Human

AND

Departments of Medicine (J.H., P.A.), Pediatrics (C.M.), Surgery (T.S.), and Research Center (J.H., P.A.), Huddinge University Hospital, Karolinska Institute, Stockholm, Sweden ABSTRACT

antagonist Y-cyanopindolol and competition experiments between this radioligand and the selective antagonists CGP-20,712-A (PI) and ICI-118551 (&) showed a 2-fold increase in the amount of fll- and & adrenergic receptors in omental as compared to SC fat cells (P < 0.02). Competition studies with the same radioligand and the nonselective pagonist isoprenaline showed no regional differences in terms of receptor affinity (Kd high 10 nM and Kd low 1 PM) or in relative fraction of receptors in the high affinity state (35%). It is concluded that an increased lipolytic sensitivity for p,- and &-agonists can be due to an increase in the amount of the two adrenoceptor subtypes in omental fat cells and thereby explain why catecholamines are more lipolytic in omental cells than in SC fat cells. (J Clin Endocrinol Metab 75: 15-20, 1992)

Catecholamine-regulation of lipolysis and /3-adrenoceptor binding isoterms were studied in human SC and omental isolated fat cells from 24 subjects undergoing elective cholecystectomy. The lipolytic sensitivity of the nonselective P-agonists epinephrine and isoprenaline as well as the selective agonists norepinephrine (PI) and terbutaline (f12) was significantly increased 5-10 times in omental fat cells. On the other hand, no regional difference in antilipolytic sensitivity was seen for the cyl agonist clonidine. No regional difference in lipolytic action was seen when measuring the effect of forskolin, (Bu),cAMP or enprofylline, which act at different postadrenoceptor steps in the lipolytic cascade. Lipolysis data showed no sex differences. A j&-pattern was seen in both regions when lipolysis dose-response curves were arranged in order of potency. Radioligand saturation experiments with the nonselective p-

D

in metabolism in different adipose tissue regions in humans may explain the higher risks of metabolic disturbances and cardiovascular diseasesassociated with abdominal obesity (l-3). Abdominal obesity seems to be associatedwith an increase more in visceral than in SC abdominal fat deposits which suggeststhat there are metabolic differences between these adipose tissue regions (2). Visceral fat has a unique possibility for interaction with the liver because it drains through the portal blood vessels. Visceral fat cells may directly deliver FFA to the liver during lipolysis; an enhancement of the latter process may cause hypertriglyceridemia and glucose intolerance (l-3). The regulation of lipolysis in man is unique as compared to other speciesbecause catecholamines are the only acutely acting lipolytic hormones in adult humans (4). In addition, catecholamines have both lipolytic (via P-adrenoceptors) and antilipolytic (via o12-adrenoceptors)effects. In both casesthe adrenoceptor receptors are coupled to adenylate cyclase, but through different coupling proteins (5). According to reviews (l-3), several studies have shown an increased lipolytic effect of catecholamines in visceral omental fat cells as compared to abdominal SCadipocytes in IFFERENCES

Received January 14, 1991. Address correspondence and requests for reprints to: Johan Hellm&-, M.D., Department of Medicine, Huddinge Hospital, S-141 86 Huddinge, Sweden. * This study was supported by the Swedish Medical Research Council, Karolinska Institute, Swedish Medical Association, Fiirenade Liv, Swedish Diabetes Association, Swedish Athletes Research Council, and the Foundations of Wiberg, Nordic Insulin, Osterman, Stohne, and Groschinsky.

man.

The

mechanisms

underlying

these

differences

are

un-

known, although we (6) and others later (7, 8) have suggested that they may be due to differences in the balance of o(- and P-receptor activity in the two fat depots. In this study we investigated the different lipolytic effects of catecholamines in SCabdominal and omental human fat cells. We first examined the effect of various agents acting on various well defined steps in lipolysis regulation and, second, measured the stoichiometric properties of /3-adrenoceptors in intact adipocytes. Materials

and Methods

Subjects The study group consisted of 24 patients who underwent elective cholecystectomy because of gallstone disease. None was on regular medication, had any known metabolic disorder, or had recently changed their food or exercise habits. One male and one female subject was overweight (body mass index > 28 kg/m2). The remaining subjects had normal body weight. They all gave their written and informed consent and the study was approved by the Ethic’s Committee at Huddinge Hospital. The group consisted of 7 males, 3 postmenopausal women and 14 premenopausal women. The age of the group was 21-74 yr (mean + SEM; 43.0 * 2.6) (men 44.2; women 42.8) and the body mass index was 19.6-30.9 kg/m’ (mean + SEM; 24.6 + 3.0) (men 25.4; women 24.2). The mean waist-to-hip ratio was 0.96 with a range from 0.84 to 1.06. Fat cell volume was similar in SC and omental fat cells (420 + 38 pl vs. 376 + 49 pl) for the whole group. General anesthesia was induced by a short-acting barbiturate and maintained by phentanyl and nitrous oxygen. The patients had fasted overnight and they received iv saline before the biopsy. The SC adipose tissue was taken from an upper paramedian incision at the beginning of surgery and the omental fat specimens were taken from the major omentum 5-10 min later.

15

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16

HELLMER

ET

JCE & M. 1992 Vo175.Nol

AL.

Adipose tissue was transported in saline to the laboratory and the preparation of isolated adipocytes, using Rodbell’s method (9) was started within 15 min after collection. The specimens were cut into fragments weighing about 5-10 mg. Adipocytes were isolated from the stroma cells by incubation with 0.5 g/liter of collagenase for 60 min in 5 ml Krebs Ringer phosphate (KRP) buffer (pH 7.4) with 40 g/liter of dialyzed BSA at 37 C in a shaking bath. The adipocytes were washed three times with a collagenase-free buffer through a silk cloth. Fat cell size was measured by direct microscopy and the mean adipocyte diameter and standard deviation were calculated from the diameter of 100 cells. Due to high adiuocvte livid content (>95%) and suherical shaue it is possible to &tima’te the mean adipocyte weight, Volume, and&cell surface area from the mean diameter. Total lipid content in the incubate was measured by organic extraction. Thereby the number of fat cells in each incubate can be calculated by dividing total lipid weight with the mean adipocyte weight. This procedure is previously described (10, 11).

binding was defined as the radioactivity measured with 1O-4 mol/liter isoprenaline present in the agonist experiments and with lo-’ mol/liter propranolol present in the antagonist experiments. Saturation experiments with a radiolabeled antagonist gives a straight line in a Scatchard plot due to its binding to G-protein coupled and uncoupled receptors with identical affinity. Displacement of a radiolabeled antagonist with an unlabeled agonist reveals a shallow biphasic curve due to the fact that binding to both coupled (high affinity) and uncoupled (low affinity) receptors are identified by the agonist. The saturation experiments were evaluated by linear regression analysis of Scatchard plots (19). Displacement curves were analyzed by a non-linear least square regression method (20). The evaluation program (LIGAND) permits a statistical comparison between a one- and a twosite model and provides the best estimates for binding isotherms. From the best fitted two-site curve it is possible to estimate the proportion of high and low affinity receptors. Only the proportion of high affinity receptors is given for simplicity reasons. The remainder is by definition the proportion of low affinity receptors.

Lipolysis

Drugs and chemicals

Adipocytes (1000-2000 cells) were incubated in 0.2 ml KRP buffer containing 40 g/liter BSA, 1 g/liter glucose, 0.1 mg/liter ascorbic acid, and various concentrations of lipolytic and antilipolytic agents. The release of glycerol was used as an index of lipolysis since glycerol, in contrast to FFA, is not reutilized in human fat cells (12). The glycerol concentration at the end of a 2-h incubation was determined in a cellfree aliquot with an ultrasensitive automatic bioluminescence method (13, 14). Glycerol release was expressed per cell surface area in order to compensate for intra- as well as interindividual differences in cell size (15). In cases where complete dose-response curves are recorded, they are compared in two respects, responsiveness and sensitivity. When responsiveness of various lipolytic agents were calculated we corrected for the difference in basal lipolysis between the two cell types by reducing all values by the basal value. In the case of clonidine (the only antilipolytic agent used), we used the relative-to-basal inhibition due to the great interindividual variability in basal lipolysis. Sensitivity was estimated graphically from the individual dose response curves as the drug concentration giving half-maximal response (ED,,). Since these values may differ by more than one order of potency within the same group, we have chosen to calculate and present such data in their logarithmic form. Furthermore, the EDs0 values are normally distributed only in their logarithmic form.

BSA (fraction V) (lot no. 63F-0748) was obtained from Sigma (St. Louis, MO). Collagenase prepared from Clostridium histolyticum was of Sigma type I. The P-adrenoceptor radioligand ‘Z51-cyanopindolol (SA, 2200 Ci/mmol) was purchased from DuPont/New England Nuclear (Boston, MA). Propranolol was from Sigma (no. P-0884) and clonidine was kindly donated by Boehringer Ingelheim (Rhein, Germany). Adenosine deaminase came from Boehringer-Mannheim (Germany). Glycerol kinase from Escherichia co/i (Sigma no. G4509) and ATP-monitoring reagent containing firefly luciferase (LKB Vallac, Turku, Finland) were used in the glycerol assay. All other chemicals were of the highest grade of purity commercially available. The same batches of hormones, collagenase, and albumin were used in all experiments,

Isolation

Radioligand

offat

cells

binding

The receptor binding studies have been described in detail (16, 17). The radioligand used was the hydrophobic and nonselective @antagonist ‘?-cyanopindolol (‘z51CYP). Freshly isolated fat cells at a concentration of about 20,00O/ml were incubated in duplicate at 37 C in 0.5 ml KRP buffer (pH 7.4) containing BSA (5 g/liter), glucose (1 g/liter), and ascorbic acid (0.1 g/liter), with and without increasing concentrations of lz5 ICYP in the saturation experiments and a fixed concentration of ‘251CYP with increasing concentrations of competing agonists or antagonists in the displacement experiments. In the saturation experiments freshly isolated fat cells were incubated for 60 min with the following concentrations of iz51CYI’; 0, 16, 31, 62, 125, 250, 500, 750, and 1000 pmol/liter. Incubations were performed in duplicate and in a third set of tubes the nonspecific binding was determined by the addition of 0.1 Fmol/liter propranolol and was estimated to be about 40%. The total amount of radioactivity added was measured in a fourth row of cell-free tubes. At the end of the incubations the cell-bound radioactivity was determined by the addition of ice-cold saline (4 ml X 3) and vacuum filtering through a Whatman GF/C filter. In the comvpetition experiments a fixed concentration of 100 pmol/ liter ‘251CYl’ was used in all incubations and the effect of increasing concentrations of the nonselective P-agonist isoprenaline, the /3, specific antagonist CGP 20,712A, or the beta* specific antagonist ICI 118,551 was studied (18). Isoprenaline was used-in 12 different concentrations (0, 1O-“-1O-4 mol/liter) whereas CGP 20.712A and ICI 118.551 were used in 10 different concentrations (0, 10-‘3-10-5 mol/liter). Nonspecific I

Statistical

analysis

Data were treated mainly as paired observations, since all experiments were performed on omental and SC tissues from the same subject, A paired t test was used for this purpose. SEM was used as a measure of dispersion, but was mostly omitted in order to increase the clarity in figures and tables. In the case of half-maximal effective drug concentration in lipolysis studies and Kd values from radioligand binding studies we have chosen to calculate data in their logarithmic form. For the comparison between the groups of men and women the Student’s unpaired t test was used. In some cases a linear regression analysis was performed.

Results Lipolysis The rate of basal lipolysis (Table 1) was higher in SC than in omental fat cells, which confirms several previous investigations reviewed in (3). Addition of adenosine deaminase to the medium increased basal lipolysis by about 50% in both depots but had no effect on the difference between regions. The maximal action of various agents on lipolysis is shown in Table 1 and Fig. 1. There were no regional differences in responsiveness for norepinephrine, forskolin (Bu)*AMP, enprofylline, or clonidine, whereas a small but significant increase in omental cells was seen for isoprenaline (P < 0.05). The findings concerning basal lipolysis and responsiveness are similar whether glycerol data are expressed per cell or per cell surface area. Complete lipolysis dose-response curves are shown in Fig. 2, where data are expressed as relative stimulation and inhibition. For forskolin, an agent known to stimulate the

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LIPOLYSIS TABLE

1. Basal

lipolysis

and lipolytic

lipolysis

Basal lipolysis with adenosine deaminase Responsiveness Norepinephrine Isoprenaline Forskolin Clonidin inhibition Relative basal value

Omental

2.5 r 0.5 (i

a

1.0 + 0.2 0

3.3 + 0.5

a

1.4 + 0.3

3.6 k 0.5 5.3 f 0.6 4.3 + 0.7

b

3.5 + 0.7 3.8 + 0.8 3.1 + 0.8 61% + 6%

74% + 3%

The Student’s paired t test was used for statistical Values are means of 14 and presented as micromoles Nm*/2 h. 10-u f SEM. a P < 0.01. *P < 0.05.

lo1 il-

Dibutyryl-CAMP

17

responsiveness

Subcutaneous Basal

IN FAT CELLS

comparisons. of glycerol per

6-

FIG. 2. Lipolysis experiments with increasing doses of norepinephrine (NE), isoprenaline (ISO), forskolin, and clonidine. The curves show the means in 14 patients. The lipolytic effects of NE, ISO, and forskolin have been calculated as relative to the maximal lipolytic effects, reduced by the value of basal lipolysis for each individual. The antilipolytic effect of clonidine is related to the maximal inhibitory effect, defined as basal lipolysis reduced by the value for maximal inhibition.

5-

TABLE

a-

Enprofyllin

7-

3-

lo--

concentrations

giving

half-maximal

effect

Subcutaneous

4-

2-

2. Drug

k -4

Group A (n = 14) Norepinephrine Isoprenaline Forskolin Clonidine Group B (n = 9) with vohimbine Norepinephrine Isoprenaline Epinephrine Terbutaline

rliil

-3 -4 -3 Drug concentration , log (mol/l)

FIG. 1. Lipolysis experiments with (Bu)zcAMP and enprofyllin. The values represent the differences between basal and stimulated lipolysis. Values are the means of 10 individual experiments + SEM. Statistical comparison with the paired t test reveals no significant differences between omental and SC cells for either drug.

catalytic component of adenylate cyclase, and for clonidine, a selective az-adrenoceptor agonist, there were no apparent differences between the omental and SC fat cells. In the case of norepinephrine, a natural catecholamine exhibiting both (Y- and b-effects, and for the P-specific agonist isoprenaline there were clear leftward shifts of the dose-response curves, indicating a higher sensitivity for catecholamines in the omental cells. A more detailed analysis based on a study of the individual half-maximal effective drug concentrations (EDso) and a comparison of them VS. the two fat cell types, confirmed the visual impression from the curves (Table 2). For forskolin and clonidine no significant difference in EDso was seen but for norepinephrine and isoprenaline there was a significant 5-fold difference in catecholamine sensitivity between the fat depots. No correlation was seen between BMI and P-adrenoceptor sensitivity in either fat depot.

The Values represents aP < *P
epinephrine = norepinephrine > terbutaline. This pattern is typical of a predominantly P1 adrenoceptormediated lipolytic action of catecholaminesin SCand omental fat cells. However, when the regions were compared, the dose-responsecurves for all P-agonists were shifted to the left in the omental region. In addition, a calculation in the individual EDs0values (Table 2), showed a significant 5- to lo-fold increasein catecholamine sensitivity in omental cells for all &agonists. Radioligand

experiments

Saturation experiments with ‘*‘ICYI’ (Fig. 4) revealed two almost parallel lines when analyzed as Scatchard plots. The mean line for omental cells was significantly shifted to the right (P < 0.01) as compared to the line obtained with SC cells. In addition, the values from both regions adapted well to a straight line in each individual experiment as well as in the mean curves; this reflects radioligand binding to a single classof receptors. An analysis of the mean maximal binding capacity as derived from the individual Scatchard plots,

-9

-8

concentration

-7

-6 , log

-5 mol/l

FIG. 3. Lipolysis dose-response curves for isoprenaline (ISO), norepinephrine (NE), epinephrine (E), and terbutaline (TER). Values are presented as relative to maximum stimulation, where the value for basal lipolysis has been subtracted. Yohimbine was added to the incubation medium in 10e4 mol/liter to abolish antilipolytic cy2 effects. Data represent the means of nine separate experiments.

showed an approximately 2 times higher P-adrenoceptor binding capacity in omental than in SCfat cells (0.3 f 0.06 lo-” amol/pm* us. 0.19 + 0.05 lo-’ amol/pm*, P < 0.02). Maximal /%adrenoceptor binding and BMI correlated in both SC(r = 0.61) and omental (r = 0.76) fat cells. In order to characterize this apparent /3-adrenergic difference more in detail we performed “‘ICYI’ competition studies with the selective antagonists CGP-20,712A (0,) and ICI 118,551 (p2) as well as with the nonselective P-agonist isopropyl-norepinephrine. When the data were analyzed with a specially designed curve-fitting data program all three agents competed with “‘ICYP according to a two-site model. The data obtained from this analysis are presented in Table 4. In a single case(Kd low for CGP) we found a significant difference between omental and SCcells but the numerical values were similar. In all other respectsand for all the three displacing agents there were no differences between the regions regarding Kd for high and low affinity binding and regarding the fraction of high affinity receptors. Taken to-

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LIPOLYSIS

0,12

y = 0,147 - 0,477x

R = 0,99

y = 0,100 -0,521x

R

q

IN FAT

0,96

T q

om

l

sl-2

ooo----l ’ 0,o 031

0,2 Bound

093

014

FIG. 4. The mean Scatchard curves from radioligand binding saturation experiments with ‘251CYP. Data are presented as means f SEM for 10 individual experiments. Plots were constructed by plotting specifically-bound (B) ligand us. bound/free (B/F) ligand. The values are compensated for cell concentration and cell size and B is expressed as a 10m5 amol/wm’. Statistical comparison of the intercepts was made using multiple regression analysis.

TABLE

4. Binding

isotherms

from

IS0

competition

experiments ICI

CGP (n = 8)

(n = 10)

(n = 9)

K, high SC Kdo;w

-8.4 -8.1

k 0.2 t 0.2

-11.0 -11.3

t 0.3 + 0.5

-11.4 -10.6

ck 0.4 + 0.7

SC

-5.8

+ 0.2

-5.9

+ 0.1 0

-6.8

+ 0.1

-5.9

+ 0.1

-6.2

+ 0.1

-6.6

k 0.1

% ‘;gh SC om

38% k 5% 36% + 5%

37% t 3% 39% + 5%

30% f 4% 30% + 3%

Competition experiments with ““I-CYP and isoprenaline (ISO), CGP-20,712A (CGP), and ICI 118,551 (ICI). Kd high and Kd low refers to the two apparent binding sites whereas percent high refers to the relative number of receptors in the high affinity state. Affinity is expressed as log mol/liter. The Student’s paired t test was used for statistical comparisons. o P < 0.05.

gether the displacement studies showed no regional differences regarding P-receptors in the balance between high and low affinity sites for agonists, agonist affinity or the fraction of PI- and &receptor sites. Discussion It is well established that catecholamines are more lipolytic in omental than in abdominal SC adipose tissue in man (68). Such differences may be located at the level of adrenoceptors, in the coupling between receptors and adenylate cyclase, in the activation of hormone-sensitive lipase by CAMP or in the breakdown of CAMP by phosphodiesterase (5). The

CELLS

19

present lipolysis data suggest that the regional variations are localized to adrenoceptors since stimulation of lipolysis at the level of adenylate cyclase or beyond that level revealed no differences between omental and SC fat cells. An additional difference is suggested by the higher basal lipolysis and the higher isoprenaline responsiveness in SC cells. These findings could be explained by a higher lipase activity in SC fat cells. The difference in basal lipolysis does not seem to depend on the endogenous adenosine content, since it was not abolished by the addition of adenosine deaminase to the incubation medium. Adenosine deaminase in itself caused only a slight increase in lipolysis in SC and omental fat cells and did not change either responsiveness or EDs0 in a full norepinephrine dose-response curve (data not shown). This suggests that adenosine had only a very moderate effect in our incubation system with very diluted fat cell suspensions (16). Like other investigators (7), we found no evidence of regional variation in the a*-antilipolytic effect of catecholamines, as judged from the data with clonidine. On the other hand, this agent is causing only a 60-70s inhibition of basal lipolysis and no experiments with stimulated lipolysis was performed in the present study. It is thus possible that some differences in oc2-receptor function may be found in omental as compared to SC adipocytes as suggested previously (7). There are several lines of evidence for regional variations in fi-adrenoceptor function in the present study. /CAdrenoceptor sensitivity was five to ten times increased in omental cells when tested with nonselective P-agonists or selective /3,- and &adrenergic agents. This suggests an increased lipolytic function in both /I-adrenoceptor subtypes in omental cells. The results with radioligand binding studies showed an increase in total P-adrenergic receptor number in omental as compared to SC fat cells. Since none of the other investigated stoichiometric properties for P-adrenoceptors differed between the regions, it is tempting to speculate that an increased catecholamine action on lipolysis in omental fat cells can be due to an increase in the amount of normally functioning &- and &adrenoceptors in these cells. It may seem odd that a relatively small difference in receptor number (2-fold) should explain a large variation in hormone sensitivity (5- to lo-fold). However, this may very well be the case. As discussed in detail (21) spare P-receptors for lipolysis are found in human fat cells. We have previously reported that under incubation conditions similar to those in the present study a small reduction in the number of cell surface P-adrenoceptors in human fat cells due to inactivation (21) or to internalization (22) causes a large change in catecholamine sensitivity. There were no apparent sex variations in the present study. Thus we observed an increased lipolytic action of catecholamines in omental fat cells in both men and women. This is somewhat in contrast to earlier findings showing regional variations in men and premenopausal but not in postmenopausal women (8). However, it is difficult to compare past and present results. The fat deposits studied were different, the age distributions in the study groups were different, and comparable sensitivity data were lacking. We have previously observed that regional differences in fl-adrenoceptor-me-

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20

HELLMfiR

diated lipolytic effects of catecholamines within the SCfat depots are very similar among men and women (17). The sex differences in this previous study was limited to a more pronounced site difference in lipolytic response to norepinephrine in women. This could be attributed to a lo-15 times lower ~2 adrenoceptor affinity in abdominal compared to gluteal adipocytes in women. No sex differences were seen for P-adrenergic lipolysis or &adrenoceptors. This latter finding was confirmed in the present study. It is well established that the lipolytic order of potency of catecholamines in human fat cells is of P1 type, although both receptor subtypes are present. The mechanismsfor this discrepancy between the presence and the function of receptor subtypes remain unclear. They may be due to variations in the coupling between receptor subtypes and lipolysis or to the existence of atypical /?-adrenoceptors(23). In this study we observed similar types of a classicalorder of potency for catecholamines in omental and SCfat cells, which suggests that there are no regional variations in this aspect of fladrenoceptor function. A previous study from our group showed that in abdominal as compared to gluteal SCadipocytes catecholamines induced a greater lipolytic response (17). This regional difference may also be largely explained by a difference in the number of /3-adrenoceptors when the data are considered together. &Adrenoceptor in visceral fat seemto be 100 times more sensitive to catecholamine stimulation than those in gluteal fat, owing to a 4 times greater number of cell surface receptor molecules; abdominal SCadipocytes have an intermediate sensitivity. Regional variations in adrenergic control of lipolysis have been shown also in the dog (24) and in the rat (25). It has been known for a long time that the lipolytic effect of catecholaminesis higher in omental as compared to SCfat cells (26). Our data suggestthat this is due at least in part to a higher sensitivity for &- and &adrenergic stimulation in the omental cells. A possible explanation for this is an increase in the number of Pi and &adrenoceptor sites in omental as compared to SCfat cells. References 1. Smith U. 1985 Regional differences in adipocyte metabolism and possible consequences in vivo. Int J Obesity 9:145-58. 2. Bjorntorp I’. 1987 Fat cell distribution and metabolism. Ann NY Acad Sci 49966-72. 3. Amer, I’. 1988 Control of lipolysis and its relevance to development of obesity in man. Diabetes Metab Rev 4:505-7. 4. Marcus C, Rhen H, Bolme I’, Arner P. 1988 Regulation of lipolysis during the neonatal period: importance of thyrotropin. J Clin Invest 82:1793-7. 5. Garcia-Saint2 J, Fain J. 1983 Adrenergic regulation of adipocyte metabolism. J Lipid Res 24:945-66. 6. Ostman J, Arner P, Engfeldt P, Kager L. 1979 Regional differences in the control of lipolysis in human adipose tissue. Metabolism 28:1198-1205.

ET AL.

JCE & M. 1992 Vol75.Nol

P, Galitzky J, Berlan M, Lafontan M. 1987 Heteroge7. Mauriege neous distribution of beta- and alfa,-adrenoceptor binding sites in human fat cells from various deposits: functional consequences. Eur J Pharmacol 17:156-65. 8. Rebuff&Strive M, Andersson B, Olbe L, Bjorntorp P. 1989 Metabolism of adipose tissue in intra-abdominal depots of non-obese men and women. Metabolism 38:453-8. 9. Rodbell M. 1964 Metabolism of isolated fat cells. I. Effects of hormones on glucose metabolism and lipolysis. J Biol Chem 239:375-80. 10. Hirsch J, Gallian E. 1968 Methods for determination of adipose cell size and cell number in man and animals. J Lipid Res 9:110-9. 11. Zinder 0, Shapiro 8. 1971 Effect of cell size on epinephrine and induced fatty acids release from isolated fat cells. J Lipid Res 12:915. 12. Arner P, Liljeqvist L, &tman J. 1976 Metabolism of mono-diacylglycerols in subcutaneous adipose tissue of obese and normal weight subjects. Acta Med Stand 200:187-94. 13. Hellmer J, Arner P, Lundin A. 1989 Automatic luminometric kinetic assay of glycerol for lipolysis studies. Anal Biochem 177:1327. 14. Lundin A, Arner P, Hellmer J. 1989 A new linear plot for standard curves in kinetic substrate assays extended above the MichaelisMenten constant: application to a luminometric assay of glycerol. Anal Biochem 177: 125-31. 15. Marcus C, Karpe B, Bolme P, Sonnenfeld T, Arner P. 1987 Changes in catecholamine induced lipolysis in isolated human fat cells during the first year of life. J ClinInvest 79:1812-9. 16. Engfeldt P, Arner P, Wahrenberg H, Ostman J. 1982 An assay for beta-adrenergic receptors in isolated human fat cells, J Lipid Res 23:715-9. 17. Wahrenberg H, Lonnqvist F, Arner I’. 1989 Mechanisms underlying regional differences in lipolysis in human adipose tissue. J Clin Invest 84:458-67. 18. Mauriege P, De Pergola G, Berlan M, Lafontan M. 1988 Human fat cell beta-adrenergic receptors; beta-agonist-dependent lipolytic responses and characterization of beta-adrenergic binding sites on human fat cell membranes with highly selective beta,-antagonists. J Lipid Res 29:587-601. 19. Scatchard G. 1948 The attractions of proteins for small molecules and ions. Ann NY Acad Sci 51:660-72. 20. Munson PJ, Rodbard D. 1980 LIGAND: a versatile computerized approach for characterization of ligand binding systems. Anal Biochem 107:220-39. 21. Arner P, Hellmir J, Wennlund A, &tman J, Engfeldt P. 1983 Adrenoceptor occupancy in isolated fat cells and its relationship with lipolysis rate. Eur J Pharmacol 146:45-56. 22. Engfeldt P, Hellmer J, Wahrenberg H, Arner I’. 1988 Effects of insulin on adrenoceptor binding and the rate of catecholamineinduced lipolysis in isolated human fat cells. J Biol Chem 263:1555360. 23. Hollenga C, Zaagsma J. 1989 Direct evidence for the atypical nature of functional beta-adrenooceptors in rat adipocytes. Br J Pharmacol 98:1420-4. 24. Taouis M, Berlan M, Montastruc P, Lafontan M. 1987 Characterization of dog fat cell adrenoceptors: variations in alpha? and betaadrenergic receptor distribution according to the extent of the fat deposits and the anatomical localization, J Pharm Exp Ther 242:1041-9. 25. Lacasa D, Agli B, Pecquery R, Guidicelli Y. 1991 Influence of overiectomy and regional fat distribution on the membranous transducing system controlling lipolysis in rat fat cells. Endocrinology 128:747-53. 26. Efendic S. 1970 Catecholamines and metabolism of human adipose tissue. III. Comparison between the regulation of lipolysis in omental and subcutaneous adipose tissue. Acta Med Stand 187:477-83.

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