Characterization of lipolytic responses of isolated white adipocytes from hamsters

458

Biochimica et Biophysica Acta, © Elsevier/North-Holland

496 (1977) Biomedical Press

458--474

BBA 28146

CHARACTERIZATION OF LIPOLYTIC RESPONSES OF ISOLATED WHITE ADIPOCYTES FROM HAMSTERS

CHRISTOPH

ROSAK

* and KARL

J. HITTELMAN

**

Department of Biochemistry, University of Massachusetts Medical School, Worcester, Mass. 01605 (U.S.A.) (Received

June

30th,

1976)

Summary 1. Lipolysis by isolated white adipocytes from hamsters, as measured by glycerol production, was stimulated by corticotropin, isopropylnorepinephrine (INE), norepinephrine, or epinephrine (EPI), in a dose-dependent fashion. 2. Lipolysis was stimulated by five inhibitors of cyclic 3',5'-adenosine monophosphate phosphodiesterase: caffeine, theophylline, 1-methyl-3-isobutyl xanthine, 1-ethyl-4-(isopropylidenehydrazine)-lH-pyrazolo-(3,4,-b)-pyridine-5-carboxylic acid ethyl ester (SQ 20009), and 4-(3,4-dimethoxybenzyl)-2-imidazolidinone (Ro 7-2956). Caffeine-stimulated lipolysis consistently attained higher rates than did hormone-stimulated lipolysis. However, when cells were stimulated by both caffeine and a hormone, lipolytic rates were consistently lower than those attained under the influence of caffeine alone. 3. Isolated white adipocytes from hamsters were sensitive to both alpha- and beta-adrenergic antagonists. The beta-adrenergic antagonist propranolol could completely inhibit norepinephrine-stimulated glycerol production. The alphaadrenergic antagonist phentolamine, on the other hand, had a biphasic effect on the cells. At 5 . 1 0 -~ M or 5 - 1 0 -6 M, phentolamine enhanced norepinephrine-stimulated lipolysis, while concentrations higher than 5 • 10 -5 M caused inhibition. 4. The effects of two different concentrations of six antilipolytic agents, prostaglandin El, nicotinic acid, phenylisopropyladenosine, 5-methylpyrazole3-carboxylic acid, adenosine and insulin, were measured. With the exception of insulin, all of these agents showed much more potent inhibition of caffeinestimulated lipolysis than of hormone-stimulated lipolysis. Insulin, in contrast, * Dr. R o s a k w a s t h e g u e s t o f t h e D e p a r t m e n t of B i o c h e m i s t r y , U n i v e r s i t y o f M a s s a c h u s e t t s M e d i c a l S c h o o l while u n d e r t h e a u s p i c e s o f a R o t a r y I n t e r n a t i o n a l F e l l o w s h i p . His p r e s e n t a d d r e s s is: J o h a n n W o l f g a n g G o e t h e U n i v e r s i t y , C e n t e r of M e d i c i n e , D e p a r t m e n t o f E n d o c r i n o l o g y , F r a n k furt]Main, Frankfurt. G.F.R. * * To w h o m r e q u e s t s f o r r e p r i n t s s h o u l d b e a d d r e s s e d .

459

showed only modest inhibition of hormone-stimulated lipolysis and virtually no inhibition of caffeine-stimulated lipolysis.

Introduction A very extensive b o d y of research accomplished over the last twenty years or so had led to the accumulation of a great deal o f information a b o u t the biochemistry and physiology of adipose tissue. By far the greatest amount of this work has been carried o u t using rats as the experimental animal. Yet that direct information which is available on the biochemistry and physiology of human adipose tissue points up some very significant differences between rat and human adipocytes, and the rat adipocyte is no longer regarded as an adequate model of the human adipocyte. For example, Rosenqvist [1--3] has shown that thyroid hormones have some effects on catecholamine responsiveness in human fat which have not been found in rat fat. Further, while it is well known that rat adipocytes display very active fatty acid and triglyceride synthesis de novo [4--6], human fat cells do not appear to synthesize fatty acids de novo to as significant a degree, but, rather, import those synthesized primarily by the liver for triglyceride synthesis [7--9]. And Burns, et al. [10] have recently discussed some major differences between human and rat fat with respect to control of lipolysis. Previous work from this laboratory has shown that with respect to cyclic AMP metabolism, the hamster adipocyte has some important characteristics which liken it to the human adipocyte. Specifically, both hamster and human adipocytes show alpha- as well as beta-adrenergic responses, while the rat adipocyte shows only beta-adrenergic sensitivity. Thus, in all three species the beta-adrenergic activity of the catecholamines has been shown to be responsible for increased intracellular cyclic AMP levels. Hamster and human adipocytes also exhibited an alpha-adrenergic response which could be shown to mediate decreases in intraceUular cyclic AMP levels which had previously been elevated by nonadrenergic (polypeptide) hormones [11--16]. The alpha-adrenergic response in hamster white adipocytes is b u t one of several ways in which hamster fat cells differ from the more commonly used rat adipocyte. Other important differences may be found in: (a) the ease and rapidity of preparation of hamster isolated adipocytes; (b) the rather narrow range of hormonal sensitivities; (c) the time course of the cyclic AMP excursion in response to a relevant agonist; (d) the magnitude of the cyclic AMP excursion in response to a relevant agonist; and (e) the effectiveness of caffeine in modifying catecholamine-stimulated increases in cyclic AMP levels. Thus, with respect to its narrow range of hormonal sensitivities and its responsiveness to alpha-adrenergic stimulation, the hamster cells more resemble human adipocytes than do rat adipocytes [14--16]. Previous work from laboratory [11,12], which called attention to some of the similarities between human and hamster adipocytes and differences between hamster and rat adipocytes, focused on cyclic AMP metabolism. It is both of interest and importance to know to what degree in the hamster these similarities to human adipocytes and differences from rat adipocytes are

460 reflected in the physiological response, lipolysis, and thus the present study was undertaken in order to describe and characterize the lipolytic responses of adipocytes isolated from hamster epididymal fat. Methods Isolated adipocytes were prepared from epididymal fat pads of 100--120 g Golden Syrian hamsters (9--12 weeks of age) obtained from Charles River Lakeview, New field, N.J. The animals were maintained at 23°C on 14 h light/ 10 h dark photoperiod and fed standard laboratory chow ad libitum. Cells were isolated in Krebs-Ringer phosphate buffer containing 3% albumin as described in detail elsewhere [11]. Schwabe et al. [17,18] have reported an effect of cell concentration on both the lipolytic and cyclic AMP responses in their studies on rat isolated adipocytes. Some experiments performed in our laboratory indicated the possibility of an effect of this nature with hamster white adipocytes, and thus it was decided to adopt throughout this study a cell concentration of 10 s cells/ml. Cell counts were made on appropriate dilutions of a concentrated stock adipocyte preparation and this stock was then diluted to 2 • 10 s cell/ml. As a control on the cell count, dilution, and pipetting procedures, a second count was made after distribution of 1-ml aliquots into the incubation vials. This second count was used to estimate the "error" of the cell dilution and distribution procedures, and yielded recoveries after the second count of 94 + 1.5%. We feel this potential error in quantifying cell number is small enough so that we are justified in expressing out data simply in terms of a cell concentration of 10 s cells/ml. In our earlier reports [11,12], data on hamster adipocytes were expressed in terms of g dry weight of cells. In order that data expressed in that manner might be compared to the cell count data obtained in this study, we have determined the correlation between cell count and dry weight, and it is shown in Fig. 1. The equation for the regression curve is y = 3089X --426. Incubations were carried out in a shaking water bath at 37°C as previously described [11]. 1 ml of the stock cell suspension (2 • 10 s cells/ml) was added to 1 ml of fresh buffer in plastic scintillation vials to yield the final concentration of 10 s cells/ml. Vials were incubated in a shaking water bath at 37°C for 20 min, appropriate additions were made, and incubation continued for desired times. All additions were made in pl volumes which did not change the final incubation volume by more than 5%. Unless noted otherwise, the incubation time was 20 min. Experiments were terminated by separating the cells from the medium by placing the entire 2 ml suspension into Pasteur pipettes plugged with glass wool. The medium was thus filtered of cells and collected in glass tubes on ice. Control experiments showed that this procedure was effective in terminating the experiment, as indicated by the stability of glycerol levels in filtered medium over a period of several hours following filtration. Glycerol was assayed by modifications of the method of Wieland [19,20]. The NADH generated by the coupled reactions of this method was determined fluormetrically in a Turner fluorometer (Model 111). In addition to a series of glycerol concentrations which were used to establish a standard curve for every

461

4xlO 5

"5 2xlO"

(..)

IxlO 5

I

O0

5LO

I0 0

I

150

Dry weight of cells (mg/ml) F i g . 1 . The relationship b e t w e e n cell c o u n t and dry w e i g h t o f cells in hamster w h i t e a d i p o c y t e prepara-

tions. Each p o i n t represents t h e m e a n +- S . E . o f 1 5 determinations. The equation for the regression c u r v e is y = 3 0 8 9 X - - 4 2 6 .

assay performed, exogenous NADH and "spiked" samples (unknowns to which known amounts of glycerol were added) were used periodically to monitor the sensitivity, precision, and reliability of the glycerol determination. We found it particularly important to use exogenous NADH and "spiked" samples to check the assay whenever new lots of commercial enzyme preparations or new glycerol standards were introduced. Crude collagenase was purchased from Worthington Biochemicals, and glycerol kinase and glycerol-3-phosphate dehydrogenase from Sigma Chemical Company. The albumin used throughout this study was bovine albumin powder, fraction V, purchased from the Metrix Division of Armour Pharmaceutical Company.Corticotropin was porcine corticotropin injection preparation from Parke-Davis Company, bioassayed by the manufacturer at 77.2 IU/mg corticotropin. Insulin was a 10 times recrystallized preparation obtained from Novo Laboratorium, Copenhangen. We gratefully acknowledge the folrowing generous gifts of special chemicals: Dr. A.J. Plummet, Ciba Pharmaceutical Company, for phentolamine; Dr. J. Pike, Upjohn Company, for prostaglandin El; Dr. R.O. Davies, Ayerst Laboratories, for propranolol; Drs. E. Westermann and K. Stock for phenylisopropyladenosine; G.D. Searle and Company for 1-methyl-3-isobutyl xanthine; Dr. C. Smith, The Squibb Institute, for SQ 20009; the Upjohn Company for 5-methylpyrazole-3-carboxylic acid; Dr. H. Sheppard, Hoffman-LaRoche, Inc., for Ro 7-2956. All other materials used were the highest quality available from commercial sources. Results

In order to ascertain the time period over which routine observations on glycerol production could be accomplished with hamster adipocytes, the time-

462 course of norepinephrine-stimulated glycerol release was first studied. Fig. 2 shows that glycerol release was linear for 30 min, after which the rate gradually declined out to 120 min. On the basis of this time-course, we chose to use a 20 min incubation period as the routine interval over which to observe glycerol release by these cells. Our previous report [11] showed that cyclic AMP levels in these cells could be elevated by catecholamines or corticotropin. Fig. 3 shows dose-response curves to norepinephrine, epinephrine, isopropylnorepinephrine and corticotropin. All of these agents evoked sigmoidal dose-response curves superimposed upon a basal level of glycerol production. To obtain an objective estimate of the EDso and related parameters, the curves of Fig. 3 were fitted by computer to a logistic function of the form ( CP ) Glycerol production = B + M Cp + K-~ as described by Parker and Waud [21]. B represents the basal level of glycerol production, M the maximal response, C the concentration of agonist, K the EDso, and p governs the slope of the curve. Table I shows the quantitative parameters yielded by this analysis. The responses rose above very consistent basal levels of glycerol production to roughly approach two maxima (see Fig. 3). The maximal rates of glycerol production stimulated by corticotropin or isopropylnorepinephrine were the highest and were nearly equal, while the maximal norepinephrine- and epinephrine-stimulated rates were somewhat lower. Further, the EDso'S are quite different for all four agonists, with almost a 100-fold difference between corticotropin and epinephrine. The EDso'S for isopropylnorepinephrine and norepinephrine were fairly close together between the cortieotropin and epinephrine values.

900 ~n o _o ×

-5 E

.S

8

=

800 70O

600

//

500 400

o o_

300

~.

200 ~00

/

/ / i

0o

/

///

I0

/

/

<

/

Agonist : 12 x 10-6M Norepinephrine

210

i

30

6'0

9LO

i

12 0

TIME (minute~) F i g . 2. T i m e - c o u r s e o f g l y c e r o l

production

by

hamster

white

adipocytes

1 . 2 " 1 0 - 6 M n o r e p i n e p h r i n e . P o i n t s are m e a n s ± S . E . o f six e x p e r i m e n t s .

in response

to stimulation by

463

300 z o

._c

e - - -o

ACTH

• ....

Isopropyl norepinephrine

•

/ ~

.~ Norepinephrine

-"

i

~'/

Epinephrine

.........

/,!.

i

/

13- ~' 12) -

-

T

i

- -

T

T

./

20C

(.9 E >.- c _j ~

BASAL O'

IO0

20' (37)

il

t/,] O

i

i

3xlO-'

I

i

3xlO -9 Agonist

Concentration

i

i

i

5xlO -7

5xlO -s

(M)

F i g . 3. G l y c e r o l p r o d u c t i o n b y h a m s t e r w h i t e a d i p o e y t e s i n r e s p o n s e t o d i f f e r e n t c o n c e n t r a t i o n s o f f o u r lipolytic agents. Basal glycerol productions at zero-time and after 20 rain of incubation are shown by bars in the left panel, with the number of experiments in parentheses. Responses to lipolytic agents are in the r i g h t p a n e l , a n d e a c h p o i n t r e p r e s e n t s t h e m e a n +- S . E . o f a t l e a s t f i v e e x p e r i m e n t s . A C T H , c o r t i c o t r o p i n .

The catecholamines and corticotropin are generally believed to stimulate lipolysis by activating adenylate cyclase. It is well known that lipolysis can also be initiated by inhibitors of cyclic adenosine-3',5'-monophosphate phosphodiesterase. Fig. 4 shows the results of experiments in which five different phosphodiesterase inhibitors were used, over a range of concentrations, to initiate lipolysis. Two widely used inhibitors, caffeine and theophylline, showed quite different dose-response curves. Although the same observed rate of lipolysis TABLE

I

QUANTITATIVE PARAMETERS DERIVEE) RESPONSE CURVES FOR CORTICOTROPIN, AND EPINEPHRINE D a t a f r o m d o s e vs. r e s p o n s e

FROM STATISTICAL ANALYSIS OF DOSE VS. ISOPROPYLNOREPINEPHRINE, NOREPINEPHRINE,

curves were fitted by computer

to a logistic function of the form R = B + M

( c P / c lj + KP), w h e r e R = t h e r e s p o n s e ( i . e . , g l y c e r o l p r o d u c t i o n ) , B = b a s a l g l y c e r o l p r o d u c t i o n , M = m a x imal glycerol production, C = concentration of agonist, K = ED$o , and p governs the slope of the curve. S e e r e f . 21 f o r d e t a i l s . Agonist

Glycerol production (nmol/2 • 105 cells per 20 min) Basal

"Slope"

function

ED50 ( r i M )

Maximal

Corticotropin

4 7 . 3 -+ 4 . 0

2 8 5 . 8 +- 1 0 . 4

2 . 1 3 -+ 0 . 4 9

1 4 . 1 -+ 2 . 8

Isopropylnorepinephrine

5 9 . 9 +- 3 . 2

2 7 8 . 8 -+ 6 . 6

1 . 4 9 -+ 0 . 2 1

1 6 2 . 6 +- 1 8 . 8

Norepinephrine

5 2 . 4 +- 3 . 8

228.1 + 9.0

1 . 2 1 +- 0 . 3 0

2 6 2 . 9 -+ 4 1 . 0

Epinephrine

47~2 +- 3 . 2

2 4 6 . 0 -+ 1 0 . 0

1 . 1 8 -+ 0 . 1 6

1 2 2 7 . 8 +- 2 2 0 . 0

464

500 Z 0

-

.;

~, Caffeine

o-

--. o Theophylline

c] . . . .

a

E3 0

R07-2956

. / xonthlne/

[-Methyl-5-isobufyl

~ =- - -e

SQ 2 0 0 0 9

./ /

0

/

200

cr$ L~ ~ >-

c

~9

/ 95%

t /

/

I00

/'

..,,// ...~"/~

BASAL ETHANOL O' 20' 20'

(19)

(19)

ill

(9)

/ / 0

L

I

l

I

I0 -s

10 -5

10-4

I0 -3

Phosphodiesterose Inhibitor Concentration (M) F i g . 4 . G l y c e r o l p r o d u c t i o n b y h a m s t e r w h i t e a d i p o c y t e s in r e s p o n s e t o d i f f e r e n t c o n c e n t r a t i o n s o f five inhibitors o f c y c l i c 3 ' , 5 ' - a d e n o s i n e m o n o p h o s p h a t e p h o s p h o d i e s t e r a s e . Basal g l y c e r o l p r o d u c t i o n at zerot i m e a n d after 2 0 rnin o f i n c u b a t i o n are s h o w n b y bars in the l e f t panel, w i t h the n u m b e r o f e x p e r i m e n t s in p a r e n t h e s e s . 95% e t h a n o l is i n c l u d e d as a c o n t r o l b e c a u s e it w a s used as the v e h i c l e for R o 7 - 2 9 5 6 and 1 - m e t h y l - 3 - i s o b u t y l x a n t h i n e . R e s p o n s e s to t h e inhibitors are in t h e right panel, and e a c h p o i n t r e p r e s e n t s t h e m e a n + S . E . o f at least five e x p e r i m e n t s .

was attained with each at 10 -3 M, there was about an order of magnitude difference with respect to the estimated EDso. The response to SQ 2 0 0 0 9 was virtually indistinguishable from that of caffeine-stimulated lipolysis. 1-methyl-3isobutyl xanthine, reported to be one of the most p o t e n t phosphodiesterase inhibitors [ 2 2 ] , evoked a peculiar response from these cells. Although it had a much lower estimated EDso than any of the other phosphodiesterase inhibitors, it elicited a maximal lipolytic rate only 50% that of theophylline or caffeine, the two other methyl xanthines. Ro 7-2956 [23] was only slightly more effective than 1-methyl-3-isobutyl xanthine in terms of the maximum response observed. The data shown in Figs. 3 and 4 suggest that in hamster adipocytes, lipolysis attained the same maximum rate when stimulated by either hormones or caffeine. However, when observations o f lipolytic rates stimulated by either or both of these means were carried out on the same cell suspension, a different pattern emerged. It was invariably found that caffeine elevated the lipolytic rate to a greater level than did hormones. Table II shows typical data comparing the effect of caffeine with that of norepinephrine or isopropylnorepinephrine. With each of the two cell suspensions n o t only can it be seen that the response to caffeine was greater than to hormones, but addition of caffeine and h o r m o n e together at maximal dosages resulted in slightly less stimulation than was attained in the presence of caffeine alone.

465 TABLE

II

EFFECT

OF NOREPINEPHRINE,

ISOPROPYLNOREPINEPHRINE,

AND

CAFFEINE

ON LIPOLYSIS

Values arc m e a n s -+ S.E. with the n u m b e r of experiments in parentheses. Glycerol production (nmol/2 - 105 cells per 20 min) Basal, 0 rain Basal, 20 min N o r e p i n e p h r i n e ( 3 • 1 0 -5 M ) Caffeine ( 1 0 - 3 M ) Norepinephrine + caffeine

28.5-+ 6.6 3 6 . 8 -+ 6 . 2 2 8 5 . 4 -+ 2 2 . 2 3 8 8 . 0 -+ 2 6 . 7 3 6 7 . 4 -+ 1 6 . 6

(5) (5)

Basal, 0 rain Basal, 20 rain Isopropylnorepinephrine Caffeine ( 1 0 - 3 M ) Isopropylnorepinephrine

2 4 . 3 -+ 5 . 8 3 1 . 4 -+ 4 . 8 3 0 5 . 4 -+ 1 3 . 2 3 3 6 . 7 -+ 1 1 . 4 275.4-+ 7.5

(5) (5) (5) (5) (5)

(3 • 10 -5 M) + caffeine

(5) (5) (5)

The stimulatory effect of caffeine was significantly greater than that of norepinephrine (P < 0.01), but there was no significant difference between the eft~ects of isopropylnorepinephrine and caffeine. This may be explained in part by the observation that isopropylnorepinephrine-stimulated lipolysis achieved a higher m a x i m u m than was attained under the influence of norepinephrine (Fig. 3); however, this would n o t explain why the combination of caffeine and hormone was not as effective as caffeine alone. These experiments led us to question whether submaximal concentrations of hormone and caffeine in combination could act synergistically in stimulating lipolysis. Table III shows the results of experiments in which 1.2 • 10 -~ M norepinephrine was used in combination with a range of caffeine concentrations. The effect seen was at best additive at caffeine concentrations of 10 -s M and 10 -~ M, and, as has already been described in Table I, higher levels of caffeine (10 -3 and 10 -2 M) in combination with norepinephrine did n o t stimulate lipolysis to the level seen with these high caffeine concentrations alone. Our initial reports on hamster white adipocytes [11,12] showed marked effects of both alpha- and beta-adrenergic antagonists on intracellular cyclic

TABLE

III

EFFECTS

OF CAFFEINE,

WITH AND WITHOUT

V a l u e s a r e m e a n s -+ S . E . f o r t h r e e e x p e r i m e n t s . v a l u e , 0 r a i n = 3 6 . 0 -+ 3 . 5 . Caffeine concentration

0 10 -5 1 0 -.4 10 -3 10 -2

(M)

NOREPINEPHRINE0

The norepinephrine

ON LIPOLYSIS

concentration

Glycerol production ( n m o l / 2 • 1 0 5 cells p e r 2 0 m i n ) --Norepinephrine

+Norepinephrine

49.3 48.8 90.3 336.3 336.5

232.2 237.5 281.4 315.6 302.4

-+ 6 . 3 -+ 3 . 2 -+ 7 . 2 +- 3 . 1 -+ 3 . 4

+- 3 . 9 +- 5 . 8 -+ 1 0 . 3 -+ 3 . 6 -+ 8 . 0

was 1.2 • 10 -6 M. Basal

466

AMP levels. We have measured the effects of two such antagonists on hormonestimulated lipolysis, and the results are shown in Table IV and Fig. 5. As expected, the beta-adrenergic antagonist propranolol was found to be a good inhibitor of norepinephrine-stimulated lipolysis in hamster adipocytes (Table IV). At a concentration of 10 -s M, lipolysis stimulated by 1.2 • 10 -6 M norepinephrine was inhibited 87%, while this propranolol concentration had only a minor effect on non-adrenergic. Corticotropin-stimulated lipolysis. At a propranolol concentration of 10 -4 M, norepinephrine-stimulated lipolysis was essentially abolished, and a major effect was also exerted on corticotropinstimulated lipolysis. Phentolamine, an alpha-adrenergic antagonist, exerted effects on non-adrenergically stimulated lipolysis of hamster adipocytes which were similar to those seen with propranolol, again especially at higher concentrations (Fig. 5). Note that as the phentolamine concentration increased, both caffeine- and corticotropin-stimulated lipolysis were progressively inhibited. Norepinephrinestimulated lipolysis, on the other hand, showed a biphasic response. At a phentolamine concentration of 5 • 10 -6 M, norepinephrine-stimulated lipolysis was enhanced by approx. 20% over the control (norepinephrine alone) level. Thereafter, as the phentolamine concentration was further increased, norepinephrine-stimulated lipolysis was rapidly inhibited. Experiments were carried out to assess the effectiveness of a number of known antilipolytic agents in inhibiting lipolysis by hamster white adipocytes. These agents were used against both hormonally-stimulated and caffeine-stimulated lipolysis, and the inhibitors exhibited quite different activities under these two circumstances. Table V shows the effects of two concentrations of each inhibitor when used against norepinephrine-stimulated lipolysis. Essentially identical results were obtained when the inhibitors wereused against corticotropin-stimulated lipol-

TABLE EFFECT

IV OF PROPRANOLOL

ON NOREPINEPHRINE

AND CORTICOTROPIN-STIMULATED

LIPOL-

YSIS

Values are m e a n s -+ S . E . w i t h t h e n u m b e r o f e x p e r i m e n t s in p a r e n t h e s e s . Basal 2 0 rain values w e r e s u b t r a c t e d f r o m all e x p e r i m e n t a l values for c a l c u l a t i o n s o f p e r c e n t inhibition. Glycerol production ( n m o l / 2 • 1 0 5 cells per 2 0 m i n ) 19.3-+ 2.0 26.9 ± 3.0 211.1 ± 10.6 233.6 + 10.0

Basal, 0 m i n Basal, 20 min N o r e p i n e p h r i n e ( 1 . 2 • 1 0 -6 M ) C o r t i c o t r o p i n (6 • 1 0 - 7 M ) Norepinephrine Norepinephrine Norepinephrine Norepinephrine

+ + + +

Corticotropin Corticotropin Corticotropin Corticotropin

propranolol propranolol propranolol propranolol

+ + + +

propranolol propranolol propranolol propranolol

(10 (10 (10 (10

(10 (10 (10 (10

-7 -6 -s -4

-7 -6 -5 -4

M) M) M) M)

M) M) M) M)

212.7 194.9 50.] 30.2

(6) (6) (6) (6)

± 9.8 (6) ± 10.5 (5) ± 1.9 ( 6 ) ± 3.2 (6)

226.8 ± 225.2 ± 201.6 ±

8.6 (5) 8.0 (6) 8.0 (6)

111.2

9.0 (6)

~

Percent inhibition

0 9 87 98 4 4 15 59

467

120

I00

.... ": -.T. "-. . . . .

g_ g

80

to

g.~

6o

o Q.

\ \

e

_o

. \

_J

40

e-

-" N o r e p i n e p h r i n e + P h e n l o l o m i n e -

~

• ....... •

\\

Coffeine +Phentolornine

.

ACTH +Phentolornine

20

0

I

I

[

I

5xlO-'

5xlO -6

5xlO -5

5xlO -4

Phentolamine Concentration (M) Fig. 5. E f f e c t o f p h e n t o l a m i n e o n t h e l i p o l y t i c r e s p o n s e o f h a m s t e r w h i t e a d i p o c y t e s to c o t t i c o t r o p i n ( A C T H ) , c a f f e i n e , a n d n o r e p i n e p h t i n e . T h e r e s p o n s e s s h o w n are e x p r e s s e d relative to t h e r e s p o n s e s m e a s u r e d in t h e a b s e n c e o f p h e n t o l a m i n e at e o r t i e o t r o p i n , c a f f e i n e , a n d n o r e p i n e p h r i n e c o n c e n t r a t i o n s of 3 • 1 0 -7 M0 1 0 -3 M, a n d 3 • 1 0 -6 M, r e s p e c t i v e l y . E a c h p o i n t is t h e m e a n -+ S.E. o f six e x p e r i m e n t s .

ysis, e x c e p t th at the percents inhibition were smaller (data n o t shown). At the lower concentrations tested, none of the inhibitors was very effective. At the higher concentrations, inhibitors were more effective, but even these inhibitions were n o t very impressive. N ot e that 2.8 • 10 .6 M prostaglandin E,, a relatively high concentration, only inhibited norepinephrine-stimulated lipolysis by

59%. When these inhibitors were used against caffeine-stimulated lipolysis, however, a completely different picture emerged (Table VI). At their lower concentrations, prostaglandin E, and nicotinic acid already almost completely inhibited lipolysis, and at the higher concentrations all the inhibitors except insulin completely blocked lipolysis. Insulin did not affect caffeine-stimulated lipolysis at either concentration tested. Adenosine is an inhibitor of lipolysis which has recently received much attention. Schwabe and his co-workers [18] have demonstrated that adenosine inhibits lipolysis in rat white adipocytes, and suggest that it may be produced as an auto-inhibitory substance by hormonally stimulated fat cells. Adenosine

468 TABLE V EFFECTS OF LIPOLYSIS

TWO

CONCENTRATIONS

OF

INHIBITORS

ON

NOREPINEPHRINE-STIMULATED

V a l u e s are m e a n s +- S.E. w i t h t h e n u m b e r o f e x p e r i m e n t s i n p a r e n t h e s e s . B a s a l 2 0 r a i n v a l u e s w e r e s u b t r a c t e d f r o m all e x p e r i m e n t a l values for c a l c u l a t i o n s of p e r c e n t i n h i b i t i o n . Glycerol production ( n m o l / 2 • 105 cells per 20 min) Basal, 0 m i n

32.6 ±

2.3 (12)

Basal, 2 0 m i n

44.3 ÷

2.5 (12)

Norepinephrine

( 1 . 2 • 1 0 -6 M )

Percent inhibition

2 3 5 . 4 -~ 9 . 9

(6)

N o r e p i n e p h r i n e + p r o s t a g l a n d i n E l ( 2 . 8 • 1 0 -9 M ) ( 2 . 8 . 1 0 -6 M )

195.7 ± 123.1 ±

4.7 3.9

(6) (6)

21 59

N o r e p i n e p h r i n e + n i c o t i n i c a c i d (3 • 1 0 . 7 M ) ( 3 ' 1 0 -5 M )

208.7 ± 170.8 +

4.1 2.5

(6) (6)

14 34

219.5 ± 189.9 ±

5.3 6.2

(6) (6)

(6) 24

204.6 ± 168.5

3.4 4.O

(6) (6)

16 35

236.6 ± 11.7 130.5 ± 12.7

(6) (6)

0 55

Norepinephrine + 5-methylpyrazole-3-carboxylic Norepinephrine + phenylisopropyladenosine

a c i d (3 • 1 0 -7 M) (3 ' 1 0 -5 M )

( 1 . 3 " 1 0 -8 M) ( 1 . 3 - 1 0 -6 M )

N o r e p i n e p h r i n e + i n s u l i n (1 p U / m l ) (1 m U / m l )

also inhibits norepinephrine-stimulated lipolysis in hamster white adipocytes (Table VII); it had no effect on basal lipolysis at any of the concentrations used (not shown). Furthermore, as with other inhibitors discussed here, adenosine was much more effective as an antilipolytic agent when used against caffeine (Table VIII) than when used against a hormone such as norepinephrine (Table VI). TABLE VI EFFECTS

OF TWO CONCENTRATIONS

OF INHIBITORS

ON CAFFEINE-STIMULATED

LIPOLYSIS

V a l u e s are m e a n s +- S.E. w i t h t h e n u m b e r o f e x p e r i m e n t s i n p a r e n t h e s e s . B a s a l 2 0 m i n v a l u e s w e r e s u b t r a c t e d f r o m all e x p e r i m e n t a l v a l u e s f o r c a l c u l a t i o n s o f p e r c e n t i n h i b i t i o n . Glycerol production ( n m o l / 2 • 105 c e l l s p e r

Percent inhibition

20 rain) Basal, 0 r a i n

2 9 . 2 -+

3.1 ( 1 2 )

Basal, 2 0 r a i n

37.9 ±

3.1 ( 1 2 )

C a f f e i n e ( 1 0 -3 M)

396.9 + 12.3 (12)

Caffeine + p r o s t a g l a n d i n E l (2.8 • 10 -9 M) ( 2 . 8 . 1 0 - 6 M)

39.4 ± 27.5-+

2.9 3.5

(6) (6)

100 100

C a f f e i n e + n i c o t i n i c a c i d (3 • 1 0 -7 M) (3 • 1 0 - s M)

7 4 . 8 -+ 32.2!

5.4 3.0

(6) (6)

90 100

2 6 5 . 9 +39.4 +

5.2 4.0

(6) (6)

37 99

341.9 ± 36.5-+

5.8 3.0

(6) (6)

15 100

401.5 ± 10.0 384.9 ± 19.7

(6) (6)

0 3

Caffeine + 5-methylpyrazole-3-carboxylic Caffeine + phenylisopropyladenosine C a f f e i n e + i n s u l i n (1 p U / m l ) (1 m U / m l )

a c i d (3 • 1 0 - 7 M) (3 • 1 0 -5 M)

( 1 . 3 • ] 0 -8 M ) ( 1 . 3 • 1 0 -6 M)

469 TABLE VII E F F E C T O F A D E N O S I N E ON N O R E P I N E P H R I N E - S T I M U L A T E D L I P O L Y S I S Values are m e a n s -+ S.E. w i t h t h e n u m b e r of e x p e r i m e n t s in p a r e n t h e s e s . Basal 2 0 rain values w e r e subt r a c t e d f r o m all e x p e r i m e n t a l values f o r c a l c u l a t i o n s of p e r c e n t i n h i b i t i o n . Glycerol production ( n m o l / 2 • 105 cells p e r 20 m i n ) Basal, 0 m i n

51.4 e

3.9 (6)

Basal, 20 m i n

56.2 •

3.9 (6)

Percent inhibition

N o r e p i n e p h r i n e (1.2 • 10 -5 M)

1 9 8 . 1 e 10.1 (6)

N o r e p i n e p h r i n e + a d e n o s i n e ( 1 0 - 9 M)

197.4 •

N o r e p i n e p h r i n e + a d e n o s i n e (10 - 8 M)

1 9 0 . 8 -+ 2.9 (6)

5

N o r e p i n e p h r i n e + a d e n o s i n e ( 1 0 -7 M)

1 9 3 . 8 -+ 5.4 (6)

4

0

2.7 (6)

N o r e p i n e p h r i n e + a d e n o s i n e (10 -5 M)

172.7-+

6.2 (6)

18

N o r e p i n e p h r i n e + a d e n o s i n e (10 -s M)

151.9+-

3.2 (6)

33

N o r e p i n e p h r i n e + a d e n o s i n e (10 -4 M)

151.9+-

5.8 (6)

33

TABLE VIII E F F E C T OF A D E N O S I N E ON C A F F E I N E - S T I M U L A T E D LIPOLYSIS Values are m e a n s +- S.E. w i t h t h e n u m b e r of e x p e r i m e n t s in p a r e n t h e s e s . Basal 2 0 rain values w e r e subt r a c t e d f r o m all e x p e r i m e n t a l values f o r c a l c u l a t i o n s of p e r c e n t i n h i b i t i o n . Glycerol production ( n m o l / 2 • l 0 s cells p e r 20 rain) Basal, 0 m i n

50.8 -+ 5.1 (6)

Basal, 20 m i n

6 3 . 4 +- 4.3 (6)

Percent inhibition

C a f f e i n e (10 -3 M)

4 4 5 . 7 + 49.3 (5)

0

Caffeine + a d e n o s i n e (10 -@ M)

4 3 4 . 7 -+ 56.1 (6)

3

Caffeine + a d e n o s i n e (10 -8 M)

4 1 9 . 5 + 47.9 (5)

7

Caffeine + a d e n o s i n e (10 -7 M)

4 5 4 . 4 -+ 63.9 (6)

0 68

Caffeine + a d e n o s i n e (10 -5 M)

1 8 3 . 9 -+ 15.4 (6)

Caffeine + a d e n o s i n e (10 -5 M)

70.3 +- 2.6 (6)

98

Caffeine + a d e n o s i n e (10 -4 M)

70.3 +

98

3.0 (6)

Discussion Very few data are available in the literature detailing the lipolytic responses of hamster white adipocytes. Some information comes from the laboratory of Rudman [24--26], but the thrust of that work was n o t specifically in the direction of characterizing lipolysis in the hamster adipocyte. Nevertheless, some comparisons between Rudman's results and ours can be drawn. Since his data are expressed as peqiv, free fatty acid released/g tissue per 2 h, the usual assumptions must be made in order to interconvert his and our data to the same units. For instance, if we assume that the release of 3 mol of free fatty acids is accompanied by the release of 1 mol of glycerol, Rudman's data indicate an epinephrine-stimulated lipolytic rate of 12 pmol of glycerol/g tissue per 2 h [25]. From Fig. 2 of the present report, we found a norepinephrine-stimulated lipolytic rate of 900 nmol glycerol/2 • l 0 s cells per 2 h. Using our Fig. 1

470 to convert from cell count to dry weight, this rate of lipolysis calculates to 13.8 pmol glycerol/g dry weight tissue per 2 h, which agrees well with the value calculated from Rudman's data. It should be noted that the hormone concentrations used in these two reports were different, with Rudman's being higher (3 • 10 -s M) than ours (1.2 • 10 -6 M). Our data shown in Fig. 3 and Table I show that the four agonists we used to stimulate lipolysis have different quantitative characteristics as reflected by their effects on lipolysis. Of some interest is the finding that the EDso for norepinephrine is about one-fifth that for epinephrine. The "order of potencies" of the beta-adrenergic component of catecholamines is usually found to be isopropylnorepinephrine > epinephrine > norepinephrine. In the case of the hamster adipocyte presented here, the order of potencies (potency = 1/EDso) isopropylnorepinephrine > norepinephrine > epinephrine, in the ratio of 7 . 5 : 4.7 : 1. At present we have no adequate explanation for this departure from the usually observed order of potencies of the catecholamines. As shown in Fig. 4, we were able to evoke significant lipolytic responses from hamster white adipocytes with all five of the phosphodiesterase inhibitors used. It is important to note here that we do not contend that these agents stimulate lipolysis solely via the inhibition of phosphodiesterase. In fact, they may well have other pertinent effects, as suggested by others, such as interference with the metabolism or action of adenosine [27] or transport of free fatty acids [28,29]. The action of 1-methyl-3-isobutyl xanthine, reportedly an extremely effective phosphodiesterase inhibitor [22], appears to be particularly complex in hamster adipocytes. Although it was the most effective of the phosphodiesterase inhibitors in stimulating lipolysis at low concentration (10 -s M), it did n o t evoke a lipolytic rate of the same magnitude as did the other methyl xanthines at higher concentrations; in fact, it showed a biphasic effect in actually becoming inhibitory at millimolar concentrations (Fig. 4). This complex interaction between 1-methyl-3-isobutyl xanthine and hamster white adipocytes serves to emphasize our poor understanding of the effects of the so-called phosphodiesterase inhibitors on cells. Our experiments with SQ 20009 show it to be virtually indistinguishable from caffeine with respect to stimulation of lipolysis by hamster adipocytes (Fig. 4). Experiments with SQ 20009 on rat adipocytes by others have given some contradictory data on this agent. For example, both Chasin et al. [30] and Allen et al. [31] report SQ 20009 to be a potent inhibitor of phosphodiesterase, which would lead one to predict that it should be a potent lipolytic agent, but Allen et al. [31] observed very little lipolytic activity on rat adipocytes. The highest rates of lipolysis we have observed with hamster adipocytes have been those attained in the presence of caffeine alone (Tables II and III). This finding is interesting for we were never able to observe any effect of caffeine alone on intracellular cyclic AMP levels, while agonists such as the catecholamines and corticotropin evoked very large increases [11]. This suggests that the increases in cyclic AMP levels observed in response to hormones were " r e d u n d a n t " to an enormous extent over that level necessary to mediate maximal lipolysis; although this functional level is apparently attained in the pres-

471 ence of caffeine, its magnitude was so small as to be below the level of sensitivity of our assay for the amounts of tissue used. We find it curious that with hamster adipocytes the combination of hormone and caffeine resulted in a lower rate of lipolysis than was observed when caffeine alone was used (Tables II and III). In other words, hormones seemed to inhibit slightly caffeine-stimulated lipolysis. This effect is apparently independent of the alpha-adrenergic activity of norepinephrine, for it was also seen with isopropylnorepinephrine, which is presumably a pure beta-adrenergic agonist. These quantitative responses differ from those seen in rat adipocytes. Fain and Wieser [32], for example, have shown that lipolysis by rat adipocytes was greater when stimulated by theophylline than by norepinephrine, b u t the combination of these two gave a still greater response. Cyclic AMP levels in hamster adipocytes are subject to both alpha- and betaadrenergic influences [ 11,12]. We have previously reported that catecholaminestimulated cyclic AMP accumulation was inhibited by the beta-adrenergic antagonist propranolol [11]. Data presented here (Table IV) show that propranolol was also a very effective inhibitor of norepinephrine-stimulated lipolysis, while it did not have a great effect on corticotropin-stimulated lipolysis, until its concentration was 10 -4 M or greater. Inhibition at such high concentrations is probably a result of other effects at the level of enzymes involved in the lipolytic process [33--35]. The alpha-adrenergic antagonist phentolamine, which we have shown enhances norepinephrine-stimulated cyclic AMP accumulation [11,12], also enhanced the lipolytic rate provided the concentration did n o t exceed 5 • 10 -~ M (Fig. 5). At higher concentrations, very marked inhibition of lipolysis occurred. This inhibitory effect has been recorded by others [36--38], and is presumably due to interference by phentolamine with enzymes of lipolysis. Recently, Stock and Thomas [38] presented evidence that under appropriate conditions phentolamine could enhance hormone-stimulated cyclic AMP accumulation in rat white adipocytes, suggesting the presence in these cells of an alpha-adrenergic receptor. However, since phentolamine augmented cyclic AMP levels elevated by isopropylnorepinephrine and corticotropin as well as norepinephrine, and was never observed to have anything but an inhibitory effect on lipolysis, it is difficult to know at present what these observations mean. Our observations on the alpha- and beta-adrenergic sensitivities of hamster white adipocytes clearly distinguish these cells from the more commonly used rat white adipocytes. As we have pointed out earlier [11], on the basis of data on cyclic AMP metabolism, the hamster adipocyte more closely resembled the human than the rat adipocyte [14--16]. This view is confirmed by our present data showing an enhancement of lipolysis in hamster adipocytes by phentolamine reminiscent of the effect of this agent on human adipose tissue [39,40]. In contrast, the more commonly used rat adipocyte does not seem to have this alpha-adrenergic sensitivity. The effects of five antilipolytic agents and glycerol production by hamster white adipocytes (Tables V and VI) show that there is a remarkable difference in the antilipolytic activity of the agents used depending upon h o w the lipolytic response is evoked. We have already reported the effects of these agents on intracellular cyclic AMP levels [12], and it was found that with the exception

472

of a high concentration of PGE~, none was able to inhibit corticotropin- or isopropylnorepinephrine-stimulated cyclic AMP accumulation by more than about 50%. Table V shows that when glycerol production is measured, roughly the same order of inhibition was seen when norepinephrine was used as agonist. Whether lipolysis was stimulated by corticotropin or norepinephrine, the most effective inhibitor of either cyclic AMP accumulation [12] or glycerol production was PGE~. In no case, however, was any agent found to inhibit completely hormone-stimulated glycerol production or cyclic AMP accumulation [11,12]. A quite different picture emerges when one tests the effectiveness of these antilipolytic agents against caffeine-stimulated glycerol production (Table VI). With the exception of insulin, all the antilipolytic agents showed complete inhibition of lipolysis at their higher concentrations, and three showed substantial inhibition even at their lower concentrations. Insulin did n o t follow the inhibitory pattern established by the other antilipolytic agents in the presence of caffeine (Table VI), yet from the data of Table V, insulin appeared similar to the other agents in the magnitude of its antilipolytic effect against norepinephrine. This apparent discrepancy may be due to different sites of action of the antilipolytic agents used. Butcher et al. [41] have shown that insulin decreased hormonally-elevated cyclic AMP levels in rat fat cells, and we have noted a small effect of this kind in hamster cells (unpublished data). Loten and Sneyd [42] have attributed this effect (in rat adipocytes) to an insulin-mediated stimulation of phosphodiesterase. Others have suggested that PGE~, nicotinic acid, 5-methylpyrazole-3carboxylic acid and phenylisopropyladenosine act by inhibiting lipolysis at a site other than phosphodiesterase, perhaps at adenyl cyclase [43--51]. It can be argued that our data of Tables V and VI support the view that insulin has a different site of action from the other antilipolytic agents used here. It is possible that under conditions where adenyl cyclase activity is presumably high and cyclic AMP levels considerably elevated, as with stimulation by norepinephrine (Table V; ref. 11), antilipolytic agents are only partially effective because they may n o t be able to completely depress cyclic AMP levels in the face of a highly active adenyl cyclase. Consequently, lipolysis is only partially inhibited (Table V). On the other hand, under the influence of caffeine, when cyclic AMP levels may be only slightly elevated (though adequate for maximal lipolysis) due to a slow, endogenous rate of cyclic AMP production which is somewhat protected due to the caffeine inhibition of phosphodiesterase, even very low concentrations of antilipolytic agents may show complete inhibition of lipolysis because they need not act against a fully active adenyl cyclase (Table VI). Consequently, as cyclic AMP production ceases completely and the cyclic AMP present is metabolized by partially inhibited phosphidiesterase, cyclic AMP levels fall to basal values, and lipolysis appears completely inhibited. Insulin would be an exception to this scheme, of course, if it acts upon phosphodiesterase rather than adenyl cyclase. Not even the high concentration of 1 mUnit/ml was capable of countering the phosphodiesterase inhibition caused by 1 mM caffeine, and insulin thus has no antilipolytic activity when caffeine is used as the lipolytic agent. Schwabe and his coworkers have presented evidence that adenosine is elab-

473

orated by hormonaUy-stimulated rat fat cells, that this nucleoside inhibits lipolysis, and that its site of action is probably at the outer surface of the plasma membrane, perhaps at adenyl cyclase [18]. While 10 -4 or 10 -s M adenosine inhibited norepinephrine-stimulated lipolysis by only 33%, essentially complete inhibition of caffeine-stimulated lipolysis was attained with 10 -s M adenosine. As with other inhibitors, this differential effect of adenosine may be better understood once we know its precise site(s) of action. In this respect it will be important to separate carefully the direct effects of these inhibitors on enzymes, such as adenyl cyclase, from more complex or indirect effects such as a competitive antagonism which may exist, for example, between adenosine and methyl xanthines, as suggested by Schwabe [27]. Although hamster and rat adipocytes appear quite similar with respect to the effects of adenosine on hormone- or methyl xanthine-stimulated lipolysis, they do differ in one respect. While basal lipolysis was markedly enhanced in rat adipocytes by adenosine [27], we have been unable to see any effect of adenosine on basal lipolysis of hamster adipocytes. Further, both hamster and rat adipocytes differ from human adipocytes in that there does not appear to be an effect of adenosine on either cyclic AMP accumulation or lipolysis in human cells [10]. Acknowledgement This work was supported by Research Grant BMS-73-06866 from the National Science Foundation. References I 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

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