ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 163, 238-245 (1974)

Regulation of Hormone Stimulation of Adipose Tissue Lipolysis

by Guanosine Triphosphate

COLIN DALTON, HARRIET HOPE, AND HERBERT SHEPPARD

Department of Cell Biology, Research Division, Hoffmann-La Roche Inc., Nutley, New Jersey 07110

Received December 21, 1973

Guanosine triphosphate (GTP) enhanced the rate of mobilization of free fatty acids from isolated rat epididymal fat cells and potentiated the lipolytic response to norepinephrine, adrenocorticotropic hormone, glucagon, and theophylline. ITP, CTP, UTP, and TTP also increased basal and norepinephrine-stimulated lipolysis but to a lesser extent than GTP. ATP differed from the other nucleotides by inhibiting norepinephrine-stimulated lipolysis. The degree of phosphorylation of the guanine was important for activity since GTP was more active than GDP which, in turn, was more active than GMP in potentiating hormone-sensitized free fatty acid mobilization. Cyclic 3’, 5’-GMP, guanine, and guanosine were inactive in this regard. Activation of lipolysis by GTP occurred immediately upon addition of the nucleotide. The lipolytic response to GTP alone or in combination with norepinephrine or theophylline was exquisitely sensitive to inhibition by prostaglandin E,. Nicotinic acid also inhibited the GTP response but to a lesser extent than prostaglandin E, and the &blocker, propranolol, had no effect. Lipolytic concentrations of GTP in combina- tion with norepinephrine increased intracellular levels of CAMP. By some as yet unknown mechanism GTP and GDP sensitized the adenylate cyclase of adipocytes to the actions of both agonists and antagonists

Guanosine triphosphate (GTP) has an obligatory role in glucagon activation of adenylate cyclase in the plasma membrane of rat hepatocyte (1, 2). Stimulation by GTP of either prostaglandin or hormone activation of adenylate cyclase has been observed in human platelets (3), frog blad- der epithelial cells (4), and beef thyroid cells (5). In a related fashion GTP pre- vented the temporal decreased response of rat erythrocyte ghost adenylate cyclase to isoproterenol (6). Harwood et al. (7) have seen both inhibitory and stimulatory ef- fects of guanyl nucleotides on fat cell adenylate cyclase.

Rodbell et al. (2) postulated that the guanyl nucleotides bind to a site on the catalytic component of the adenylate cy- clase that differed from the hormone bind- ing site. These authors suggested that GTP has an intracellular regulatory function whereby ATP becomes more available for its catalytic conversion to cyclic 3’) 5’-

AMP. The activity of guanyl nucleotides on other sites on the membrane have not been examined. We have, therefore, elected to study the response of rat isolated fat cells to added GTP and related sub- stances.

MATERIALS AND METHODS

Epididymal fat pads were excised from Charles River rats weighing between 190 and 210 g. Adipo- cytes were separated from the pads by means of a crude collagenase as reported by Rodbell (8). The fat cell suspension was incubated in plastic vials con- taining Krebs-Ringer bicarbonate buffer (9), but with half the suggested concentration of calcium ion and 4% bovine albumin, pH 7.4, for 1 hr (unless otherwise stated) at 37°C in a Dubnoff metabolic shaker. Drugs and hormones were dissolved in the Ringer/albumin solution. The incubation volume was 1 ml. PGE2’ was dissolved in ethanol and diluted to working concen- trations with Ringer/albumin solution. An appropriate ethanol control was run in these PGE, experiments.

‘Abbreviations: ACTH, adrenocorticotropic hor- mone; FFA, free fatty acids; PGE?, prostaglandin E2.

238 Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.

ACTIVATION OF LIPOLYSIS BY GTP 239

For free fatty acid determination the reaction was terminated by adding 3 ml phosphate buffer (pH 6.2) to each flask, followed by 20 ml chloroform. The vials were capped, shaken for 10 min, allowed to stand in order to separate the two phases, and the upper aqueous layer was aspirated. Free fatty acid analysis was conducted directly on the chloroform extract by the autoanalyzer procedure of Dalton and Kowalski (10). Aliquots of the chloroform extract used for triglyceride determination were evaporated to dryness under N, and redissolved in 10 ml of aldehyde-free isopropanol, and analyzed by the automated fluoro- metric method of Kessler and Lederer (11). The rate of lipolysis is expressed as pequivalents of free fatty acids released per gram of fat cell triglyceride per hour.

Cyclic AMP Determination In experiments in which CAMP was determined the

reaction was stopped after 10 min since preliminary experiments confirmed the observation of Butcher et al. (12) that maximal levels of CAMP were obtained within 10 min of incubation in the presence of lipolytic hormones or theophylline. Five tenth-milli- liter aliquots of the fat cell suspension were sonicated with ice cold 6% trichloroacetate for 10 min, cen- trifuged at 3000 rpm for 30 min in an International Refrigerated Centrifuge Model PR-6 and the superna- tant was washed three times with water-saturated ethyl ether. Finally the samples were evaporated to dryness at 60°C under nitrogen and stored at -40°C. The samples were dissolved in 0.5 ml acetate buffer, pH 6.2, prior to analyses for CAMP using the Brown et al. (13) modification of the protein-binding assay described by Gilman (14).

The substances used in this study were: guanosine- 5’.triphosphate (sodium salt), guanosine-5’.diphos- phate (sodium salt); guanosine-5’.monophosphate (sodium salt), guanosine-3’, 5’.cyclic monophosphate (sodium salt), guanosine (anhydrous), and guanine, Sigma Chemical Co. The sodium salts of adenosine- 5’-triphosphate (lot no. 188A), uridine-5’-triphos- phate (lot no. 17002), inosine-5’.triphosphate (lot no. 026001), cytidine-5’.triphosphate (lot no. 112001!, and thymidine-5’-triphosphate (lot no. 258001), PL. Biochemicals; propranolol (Inderal) L-iso- propylamino+(l-naphthyloxy)-2-propanol HCl, Im- perial Chemical Industries, Ltd. Wilmslow, England; norepinephrine bitartrate, Winthrop Laboratories, Inc. New York, NY; collagenase, Worthington Bio- chemicals, Freehold, NJ; Synacthen, Ciba Phar- maceutical Co., Summit, NJ; glucagon (lot no. 802097) Calbiochem., San Diego, CA 90054. Other materials used were supplied by the Chemical Re- search Division of Hoffmann-La Roche Inc. All the chemicals used, which were of the highest grade available, were used without further purification. The collagenase and phosphate nucleotides were stored as

dry powders in a desiccator at -20°C until immedi- ately prior to use.

RESULTS

Figure 1 shows that basal and norepi- nephrine-stimulated mobilization of free fatty acids (FFA) from rat isolated fat cells is enhanced by the presence of 250 FM

GTP. Other triphosphate nucleotides, added at the same concentration, also enhanced lipolysis but to a lesser extent than the guanosine compound. ATP had little effect on basal lipolysis and distinctly inhibited norepinephrine-stimulated lipol- ysis.

GTP also potentiated the effect of sub- maximal concentrations of other lipolytic agents on adipose tissue lipolysis (Fig. 2). The lipolytic response to glucagon and ACTHlm2’ (Synacthen), which activate the adenylate cyclase in adipose tissue, and to theophylline, which is presumed to work by inhibiting adipose tissue phosphodiester- ase, were markedly potentiated by GTP.

A dose-response curve for the effect of GTP on mobilization of FFA produced by low concentrations of norepinephrine is shown in Fig. 3. The effect of other guanine derivatives on basal lipolysis and norepi- nephrine-stimulated lipolysis is recorded in Table I. The degree of phosphorylation in the molecule clearly alters its lipolytic potency. The triphosphorylated molecule is more active than the diphosphate which in turn is more active than GMP as a stimulator of lipolysis or as a potentiator of norepinephrine-stimulated lipolysis. Gua- nosine was found to be inhibitory of basal lipolysis, as reported by Davies (151, and had no effect on norepinephrine-induced lipolysis. Dole (16) had previously recorded a stimulatory effect of guanine in rat epi- didymal fat pads and here we found a slight stimulatory response in isolated fat cells. Norepinephrine-induced lipolysis, however, was not potentiated by guanine. Cyclic 3’,5’-GMP did not activate lipol- ysis.

A time-course experiment in which the effect of GTP on lipolysis was studied in the presence and absence of norepineph- rine revealed that the stimulation of FFA release due to GTP occurred immediately

240 DALTON, HOPE, AND SHEPPARD

NOREPINEPHRINE-INDUCED LIPOLYSIS

140

120

100

SO

60

40

ATP “TP ITP CTP TTP

CONTROL GTP ATP UTP ITP CTP TTP

FIG. 1. Effect of nucleotide triphosphates on basal and norepinephrine-induced lipolysis in rat isolated fat cells. Rate of lipolysis represents the mean of four to six flasks per condition. Norepinephrine, lo-’ M;

concentration of the nucleotides, 2.5 x lo-’ M.

160- GTP (OlmMl

Basal Glucoqon ACTH I

Theo

FIG. 2. Effect of GTP on glucagon-, ACTH-, and theophylline-induced lipolysis in rat isolated fat cells.

[Rate of lipolysis represents the mean of four to six flasks per condition. Glucagon, 0.5 fig/ml; ACTH’-z’ (Synacthen) 50 rig/ml; theophylline, 0.1 mM.

after addition of GTP (Fig. 4). These data strongly suggest that the phenomenon is a true activation rather than an induction effect dependent on new protein synthesis.

In order to gain knowledge of the mecha- nism of action; GTP lipolytic activity in the presence of some known blocking

; 4ot< , ] s

d rc, con::n;lro+lo”‘0-3 ‘O- 2

FIG. 3. Concentration-dependent effect of GTP on norepinephrine-induced lipolysis. Each point repre- sents the mean of four flasks per condition, norepi- nephrine, lo-’ M.

agents was studied. The adrenergic p- blocker, propranolol, (1 PM) effectively blocked the norepinephrine response in fat cells and also partly inhibited the com- bined response of norepinephrine and GTP (Table II). Propranolol did not block the lipolytic activity of theophylline, GTP, or

ACTIVATION OF LIPOLYSIS BY GTP 241

TABLE I

EFFECT OF GUANINE DERIVATIVES ON BASAL AND NOREPINEPHRINE-INDUCED LIPOLYSIS IN RAT

ISOLATED FAT CELLS~

Additions Rate of Lipolysis

Basal

Control 5.1 * 0.9 GTP 37.1 * 1.4’ GDP 8.7 * 1.3 GMP 5.9 i 1.0 Guanosine 1.9 * 1.3 Guanine 9.8 * 0.9” Cyclic 3’) 5’.GMP 7.9 i- 0.6

Norepi- nephrine

29.4 i 1.1 149.7 * 4.7’ 72.2 * 1.7’ 50.0 zt 0.8’ 25.9 * 1.4 27.4 * 0.5 33.0 * 1.1

’ The rate of lipolysis represents the mean of four or six flasks per condition expressed as microequivalents of free fatty acids released per gram of fat cell triglyceride per hour * standard error of the mean. Concentration of guanine derivatives was 200 pM and concentration of norepinephrine was 0.5 pM. The significance of the difference due to additions, *, P < 0.05; c, P < 0.001.

140

i

120

t

40

I I I I I

5 IO 15 20 TIME IN MINUTES

FIG. 4. Time response of the effect of GTP on the activation of basal and norepinephrine-induced lipol- ysis. Concentration of the agents added individually or in combination; norepinephrine, 5 x 10m7 M; GTP, 10-J M.

the combination of GTP and theophylline. These experiments clearly eliminate the fat cell /3-adrenergic receptor as the site of action of GTP.

Prostaglandin E, is a known antilipolytic

agent (17). We found PGE, (0.28 PM)

caused a large blockade of theophylline- stimulated lipolysis and a lesser blockade of norepinephrine-stimulated lipolysis. The lipolytic response of GTP alone or in combination with theophylline or norepi- nephrine was very significantly blocked by PGE, (Table III). PGE, inhibition of FFA mobilization was studied further by explor- ing the inhibitory response curves of PGE, versus norepinephrine-, theophylline-, and GTP-stimulated lipolysis (Fig. 5). GTP was clearly more sensitive to inhibition by PGE, than was theophylline or norepi- nephrine. Their apparent Ki values were 7, 15, and 80 nM, respectively. PGE, com- pletely reversed the GTP response at 56 nM and at higher concentrations of PGE2, values below the basal rate of FFA release were recorded.

Similarly nicotinic acid is more effective as an antilipolytic agent versus theophyl- line-stimulated lipolysis than it is versus norepinephrine-stimulated lipolysis (18). Our experiments show nicotinic acid weakly blocked the norepinephrine- induced lipolysis but strongly inhibited the response due to theophylline, GTP, and the combination of norepinephrine or theophylline and GTP (Table IV).

TABLE II

EFFECT OF PROPRANOLOL ON THE LIPOLVTIC ACTKITY OF GTP, NOREPINEPHRINE, AND THEOPHYLLINE IN RAT

ISOLATED FAT CELLS~

Addit ions Rate of Lipolysis

Without With propranolol propranolol

Basal 5.0 * 0.7 7.2 i 0.9 GTP 13.4 * 2.3 12.1 * 0.4 Norepinephrine 33.4 * 0.8 18.3 * 0.6’ Norepinephrine + GTP 84.5 * 7.3 56.6 * 3.v Theophylline 304 * 1.7 311 * 2.8 Theophylline + GTP 313 * 5.0 313 i 4.9

“The rate of lipolysis represents the mean of four flasks per condition expressed as microequivalents of free fatty acids released per gram of fat cell triglyc- eride per hour f standard error of the mean. Concen- tration of agents added individually or in combination were: GTP, 10 4 M; norepinephrine, 5 )i 10m7 M;

theophylline, 1 mM, and propranolol, 1 PM. The significance of the difference due to propranolol, b, P < 0.05; c, P < 0.001.

242 DALTON, HOPE, AND SHEPPARD

TABLE III

EFFECT OF PROSTAGLANDIN-E,, ON THE LIPOLYTIC

ACTIVITV OF GTP. NOREPINEPHRINE, AND

THEOPHYLLINE IN RAT ISOLATED FAT CELLS”

Addit ions

Control GTP Norepinephrine Norepinephrine + GTP Theophylline Theophylline + GTP

Rate of Lipolysis

m

7.8 i 1.7 14.5 * 1.9 45.2 * 2.0 122 * 2.7 285 + 4.6 301 * 5.7

9.6 i 2.2 6.9 zt 0.3b

29.7 * 0.7’ 44.9 i 2.8’ 27.6 i 1.4’ 28.6 + 1.0’

a The rate of lipolysis represents the mean of four flasks per condition expressed as microequivalents of free fatty acids released per gram of fat cell triglyc- eride per hour * standard error of the mean. PGE, 2.8 x lo-’ M was added in 0.10 ~1 ethanol. all flasks received an equal amount of ethanol. Concentrations of the other agents added individually or in comhina- tion were: GTP, 0.1 mM; norepinephrine 0.5 pM,

theophglline 1 mM. The significance of the difference due to prostaglandin, ‘, P < 0.05; ‘, P < 0.001.

The effect of GTP and GTP in combina- tion with norepinephrine on cyclic 3’) 5’- AMP levels in adipocytes was determined. Intracellular CAMP levels were elevated 4-fold in these experiments by a lipolytic concentration of norepinephrine (Table V) as is consistent with published observa- tions (12). GTP by itself caused a slight, nonsignificant increase in CAMP but in combination with norepinephrine GTP greatly increased intracellular levels of CAMP. This result may explain the mecha- nism of action of GTP as a lipolytic agent. There is a fairly good relationship between CAMP levels and the rates of mobilization of FFA observed under the same incuba- tion conditions. This relationship breaks down in the case of the extremely high concentrations of CAMP generated by the combination of two lipolytic agents be- cause, as has been explained by Butcher et al. (12), the concentration of CAMP far exceeds that required for maximal lipol- ysis.

An inhibition of fat cell cyclic AMP phosphodiesterase by GTP could explain the potentiation of hormone-sensitized li- polysis as has been demonstrated with theophylline (19) and 4-( 3,4, -dimethoxy-

benzyl)-2-imidazolidinone (20). GTP, how- ever, was tested directly for this effect on both 105,000 g supernatant and fat cell membranous phosphodiesterase prepara- tions, using the method of Weiss et ~2. (21), and was found to have neither inhibitory nor stimulatory activity.

DISCUSSION

Previous studies have demonstrated an enhancing effect of GTP on glucagon (1,2), prostaglandin E, (3, 5), epinephrine (7, 22), thyrotropic hormone (5), adrenocor- ticotropic hormone (7), oxytocin (4), and. isoproterenol (6) activation of adenylate cyclase in various tissues. Our work has extended these observations to include an enhancement of the effects of norepineph- rine and theophylline and, of even greater significance, an action on an intact cell. Our data support the concept (2, 7) that GTP acts on the membranes in such a way as to make them more sensitive to the various stimulants and inhibitors.

An elevation of fat cell CAMP caused by lipolytic concentrations of GTP appears to

-201 PGE2

FIG. 5. Inhibitory concentration response curves of PGE, vs norepinephrine-, theophylline-, and GTP- induced lipolysis. Each point represents the mean of four or eight flasks per condition. (0) GTP, lo-* M,

(x) norepinephrine, 5 x 10ml M, and (0) theophylline, lo-’ M.

ACTIVATION OF LIPOLYSIS BY GTP 243

TABLE 11~

EFFECT OF NICOTINI~ ACID ON THE LIPOLVTIC ACTWTI OF GTP, NOREPINEPHRINE. AND THEOPHYI.LINE IN RAT

ISOLATED FAT CELW

Additions Rate of’lipolysis

Without With nicotinic nicotinic

acid acid

Control 9.:1 * 1 .o 7.7 f 0.7

GTP 16.*5 + 0.6 9.4 i 0.8’ Norepinephrine x1.0 k 0.9 23.0 t 1.2’ Norepinephrine + GTP 76.7 i 3.2 44.5 * 2.0” Theophylline 213 I 2.5 95.6 z 6.9” Theophylline + GTP 207 + 6.” 118 i 4.7”

” The rate of lipolysis represents the mean of four flasks per condition expressed as microequivalents of free fatty acids released per gram of fat cell triglyc- eride per hour + standard error of the mean. Concen- trations of the agents added individually or in comhi- nation were. GTP. 0.1 mM; Korepinephrine. 0.5 FM; theophylline. 1 mM; and nicotinic acid. I PM. The significance of’ the difference due to nicotinic acid h. P < 0.05: ‘. P < 0.01 and ‘, P < 0.001.

account for the lipolytic mechanism of action of GTP. CAMP then activates a CAMP-dependent protein kinase which in turn activates a hormone-sensitive lipase responsible for the hydrolysis of depot triglycerides (23, 24). We do not know how the intracellular levels of CAMP are ele- vated by GTP. Our data conforms to the concept that GTP may act in an allosteric fashion on a regulatory component of the adenylate cyclase and thereby increase the coupling between the hormone receptor and the catalytic unit (2). Whatever the mechanism, guanine nucleotides appar- ently have access to these sites from the outside of the cell. Although at first glance this might seem at variance with observa- tions of GTP action on isolated liver mem- branes, similar membranous preparations are known to have a tendency to form vesicles of various sizes rather than remain in extended or open conformations.

Erythrocyte (6) and platelet (3) prepara- tions were prepared by hypotonic lysis and their vesicular nature was alluded to by the respective authors. Therefore, the addition of GTP in those cases, as well as with the intact fat cell, required that the nucleotide proceed to its site of action from the

outside of the vesicle. In both cases, how- ever, the medium used in the assay was hypotonic and presumably the GTP, as well as the substrate ATP, could move through the membrane into the vesicle. One has to entertain the notion that with our adipocytes, permeability had been in- creased during their preparation with the collagenase/protease mixture. In any event, the GTP is apparently capable of effecting some alteration in the membrane which increases the responsivity of the adenylate cyclase.

The observations by Cryer et al. (25) that GTP and other nucleotides inhibited adenylate cyclase in fat cell membranes are not consistent with the data reported here but may be explained by the experi- ments of Harwood et al. (7). The latter authors reported two independent effects of guanyl nucleotides on basal and hor- mone-stimulated fat cell adenylate cyclase activity. GTP inhibited basal activity and, by an apparently different mechanism, GTP converted the membranous enzyme to a thermally stable state characterized by increased sensitivity to hormonal stimula- tion. Inhibition by GTP may be the result of nonspecific binding to fat cell adenylate cyclase, similar to that reported by Birn- baumer and Pijhl (26) for guanyl nucleo- tide binding to liver plasma membranes. Birnbaumer and Piihl (26) showed that GTP stimulated dissociation of bound la- beled glucagon from liver membranes under conditions which do not selectively affect the glucagon stimulation of adenyl

TABLE \

EFFECT OF GTP ON CYCIJC 3’, 6’.AMP 1,~wt.s IN RA.I Isor ATFD FAT CFI I.S” 1 I ,11

Addit ions picomolesig 1 SE

None 261 i 17 GTP :3i7 + 48 Norepinephrine 1009 f 4,5,” Norepinephrine - GTP 9597 : 776”

’ Fat cells were preincuhated f’or 20 min at room temperature then incubated for 10 min at 37°C in the presence of GTP, 0.5 mM, norepinephrine. 05 I’M or their combination. Values represent the mean of’ four flasks per condition. The significance of the dift’erence due to the additions, ‘. P < 0.001.

244 DALTON, HOPE,

cyclase activity, suggesting that the effects of purine nucleotides on hormone binding and on hormone stimulation may be un- related phenomena.

GTP regulation of lipolysis is probably not associated with endogenous generation of cyclic GMP since nucleotides other than GTP showed appreciable activity and ad- dition of cyclic 3’) 5’-GMP did not activate lipolysis. The failure of the nucleosides and bases to increase fatty acid mobilization indicates that the dephosphorylated prod- ucts are of little importance. Thus, what- ever the mechanism, GTP must be operat- ing through the phosphorylated form. A mechanism involving cyclic nucleotide phosphodiesterase is also unlikely since GTP, GDP, and GMP at lipolytic concen- trations did not inhibit the fat cell phos- phodiesterase.

Occupancy of the /3-adrenergic receptor has also been eliminated as a possible site of action because propranolol, at adrener- gic blocking concentrations, failed to in- hibit the GTP response. GTP-stimulated lipolysis was, however, very sensitive to inhibition by PGE, and nicotinic acid. In these respects GTP acts more like the- ophylline than norepinephrine. Although this seems to contradict the observation that GTP is not an inhibitor of phospho- diesterase, there is appreciable evidence suggesting that theophylline may stimulate FFA mobilization by mechanisms other than inhibition of the fat cell phosphodies- terase (20, 27, 28). Perhaps both agents are acting through a control of ion move- ments in the membrane. Addition of high concentrations of PGE, to GTP-activated fat cells resulted in lipolytic values below the basal rate. This response cannot be ex- plained at the moment but may represent an effect against an endogenous stimula- tory component in lipolysis regulation which is not nomally available to inhibi- tion by PGE, but is exposed (or activated) by the presence of GTP.

Thus, the original observations of Rod- bell et al. (1, 2) concerning the action of GTP in the liver plasma membrane prepa- ration have been extended to include the isolated functioning adipocyte. The possi-

AND SHEPPARD

bility that GTP is acting on the “outside” of the membrane is strengthened. GTP- mediated changes in the cell apparently resulted in increased sensitivity to both lipolytic agonists and antagonists.

ACKNOWLEDGMENT

The authors thank R. Scism and L. Martikes for technical contributions and M. Johnson for typing the manuscript.

1.

2.

3.

4.

5.

6.

7.

8. 9.

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12.

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15. 16. 17.

18.

REFERENCES

RODBELL, M., KRANS, H. M. J., P~~HL, S. L., AND BIRNBAUMER, L. (1971) J. Biol. Chem. 246, 1861-1876.

RODBELL, M., BIRNBAUMER, L., P~HL, S. L., AND KRANS, H. M. (1971) J. Biol. Chem. 246, 1877-1882.

KRISHNA, G., HARWOOD, J. P., BARBER, A. J., AND JAMIESON, G. A. (1972) J. Biol. Chem. 247, 2253-2254.

BOCKAERT, J., ROY, C., AND JARD, S. (1972) J. Biol. Chem. 247, 7073-7081.

WOLFF, J., AND COOK, G. H. (1973) J. Biol. Chem. 248, 350-355.

SHEPPARD, H., AND BURGHARDT, C. R. (1972) The Role of Membranes in Metabolic Regulation (ed. Mehlman, M.), pp. 385-409, Academic Press, New York.

HARWOOD, J. P., Low, H., AND RODBELL, M. (1973) J. Biol. Chem. 248, 6239-6245.

RODBELL, M. (1964) J. Biol. Chem. 239, 375-380. UMBREIT, W. W., BURRIS, R. H., AND STAUFFER, J.

F. (1957) Manometric Techniques, 3rd Ed., p. 149, Burgess, Minneapolis.

DALTON, C., AND KOWALSKI, C. (1967) Clin. Chem. 13, 744-751.

KESSLER, G., AND LEDERER, H. (1966) in Automa- tion in Analytical Chemistry (Skeggs, L. T., ed.) pp. 341-346, Mediad Inc., New York.

BUTCHER, R. W., BAIRD, D. E., AND SUTHERLAND, E. W. (1968) J. Biol. Chem. 243, 170551712.

BROWN, B. L., ALBANO, J. D. M., EKINS, R. P., AND SGHERZI, A. M. (1971) Biochem. J. 121,561-562

GILMAN, A. G. (1970) Proc. Nat. Acad. Sci. USA 67, 305-312.

DAVIES, I. (1968) Nature (London) 218, 349-352. DOLE, V. P. (1961) J. Biol. Chem. 236,3125-3130. STEINBERG, D., VAUGHAN, M. NESTEL, P. J., AND

BERGSTROM, S. (1963) Biochem. Pharmacok 12, 764-766.

DALTON, C., QUINN, J. B., CROWLEY, H. J., AND

MILLER, 0. N. (1971) in Metabolic Effects of Nicotinic Acid and its Derivatives (Gey, K. F., and Carlson, L. A., Eds.), pp 331-339, Hans Huber, Bern.

ACTIVATION OF LIPOLYSIS BY GTP 245

19. HYNI~ S., KRISHNA, G., AND BRODIE, B. B. (1966) J. Phurmacol. Exp. ‘I’her. 153, 90-96.

20. DALTON, C. QUINN, J. B., BIJRGHARDT, C. R., AND SHEPPARD, H. (1970) J. Pharmacol. Exp. Thu. 173, 270-276.

21. WEISS, B., LEHNE, R., AND STRADA, S. J. (1972) Anal. Biochem. 45, 222-235.

22. LERAY, F., CHAMBAUT, A. M., AND HANOUME, J. (1972) Biochem. Biophys. Res. Commun. 48, 138551391.

23. CORBIN, J. D., AND KREBS, E. G. (1969) Biochem.

Biophys. Res. Commun. 36, 328-336. 24. HUIXJNEN, J. K., STEINBERG, D., ANDMAYER, S. E.

(1970) Proc. Nat. Acad. Sci. USA 67, 290-295. 25. CRYER, P. E., JARETT, L., AND KIPNIS, D. M. (1969)

Biochem. Biophys. Acta 177, 586-590. 26. BIRNBAUMER, L. AND PGHL, S. L. (1973) J. Biol.

Chem. 248, 2056-2061. 27. MCNEILL, J. H., MASSA& M., AND BRODY, T. M.

(1969) J. Pharmacol. Exp. Ther. 165, 234-241. 28. SCHWABE, U., and EBERT, R. (1972) Neunyn-

Schmiedebergs Arch. Pharmakol. 274, 287-298.