Life Sciences, Vol. 32, pp. 1515-1522 Pergamon Pres Printed in the U.S.A.

STIMULATION OF CYCLIC AMP AND LIPOLYSIS IN ADIPOSE TISSUE OF NORMAL AND OBESE AVY/a MICE BY LY79771, A PHENETHANOLAMINE, AND STEREOISOMERS

Terence T. Yen, Marilynn M. McKee, Nancy B. Stamm, and Kerry G. Bemis

L i l l y Research Laboratories, El i L i l l y and Company, Indianapol is, IN 46285

(Received in final form December 2], 1982)

Summary

The st imulat ion of cyc l ic AMP and l i po l ys i s by LY79771, a phenethanolamine ant iobesi ty compound, and i t s 3 stereo- isomers in adipose tissue of obese viable yellow mice and normal mice were studied. Both a c t i v i t i e s were stereo- specif ic with LY79771, the R,S isomer, and LY79730, the R,R isomer, being more potent than LYI03085, the S,S isomer, and LYI03672, the S,R isomer. Propranolol, a nonspecific B-antag- onist , completely inhibi ted the elevation of cycl ic AMP and l i po l ys i s whereas atenolol, a speci f ic 81 antagonist, inhib i ted the elevation of cycl ic AMP but did not completely i nh ib i t l i p o l y s i s . These f indings indicate that the eleva- t ion of cyc l ic AMP was mediated by the BI- receptor whereas the st imulat ion of l i po l ys i s was mediated by both the 81 and B2 receptors. The adipose tissue of the obese viable yellow mice responded to these compounds less than that of the normal mice.

Compound LY79771 reduced weight or decreased weight gain of genet ica l ly obese rats and mice, gold thioglucose obese mice, and obese beagles without af fect ing the i r food consumption ( I ) . A decrease in body fat accounted for most of the weight change caused by LY79771. Since LY79771 has two asym- metric centers, a total of 4 stereoisomers is avai lab le (Fig. I ) . This communication compares the effects of LY79771 and i t s stereoisomers on cyc l ic AMP and l i po l ys i s in adipose tissue of normal and viable yellow obese mice in an attempt to decipher the mechanism of these compounds.

Materials and Methods

Mice. Mature, male, inbred viable yellow obese mice (VY/WfL-AvY/a) and normal mice (VY/WfL-ala) from our o~ colony were used. At the time of the experiments, the-mTce were at least 5 months old. They were housed in transparent p last ic cages, 2 to 4 mice in each cage. The ambient temperature of the animal room was maintained at about 25°C. The l igh ts in the room were on for 12 hours per day. Water and Purina Chow 5008 containing 6.5 percent fa t were avai lable ad l ib i tum.

Compounds LY79771, LY79730, LYI03672 and LYI03085 were synthesized by Drs. J. Mi l l s and K. K. Schmiegel of our Research Laboratories. Sources for other chemicals were: theophyl l ine, Matheson, Coleman and Bel l ; (~)-propranolol HCl, Sigma; and atenolol , courtesy of Stuart Pharma- ceut icals Division of ICl United States, Inc.

0024-3205/83/131515-08503.00/0 Copyright (c) 1983 Pergamon Press Ltd.

1516 LY79771 Stimulation of cAMP and Lipolysis Vol. 32, No. 13, 1983

OH 1. . / ~

-CH -CH2-NH-~H *-CH2-CH2- ~ I ~ - O H

CH 3

HCI

C* C *~ 79771 R 79730 R R

I03085 S S 103672 S R

FIG. 1

Structures of LY79771 and isomers

In v i t r o cyc l ic AMP formation. The formation of cyc l ic AMP in adipose t issue in v i t r o was measured by the method of Dehaye et al. (2). Three hundred to 600 mg of epididymal adipose t issue was f i r s t incubated at 37°C for 30 min in Krebs-Ringer bicarbonate buf fer , pH 7.4, containing 3 percent f a t t y -ac id - f ree bovine albumin, f rac t ion V (Sigma), and 10 -3 M theophyl l ine. The tissue was then transferred to fresh Krebs-Ringer bicarbonate buffer containing 3 percent f a t t y -ac id - f ree bovine albumin, f rac t ion V, IO-3M theophyl l ine, and the specif ied concentrations of chemicals and incubated at 37°C for 7 min under an atmosphere of 95 percent 02 and 5 percent CO 2. At the end of the 7 min incubation, the t issue was homogenized in 0,3 N perchloric acid and centrifuged at 20,000 x G. The cyc l ic AMP in the supernatant was eluted from Dowex columns and measured with the cyc l ic AMP radioimmunoassay k i t supplied by New England Nuclear. A 3H-cyclic AMP marker was used to determine percent recovery.

In v i t r o l i p o l y s i s . About I00 mg of epididymal adipose t issue was incubated in Krebs-Ringer bicarbonate buf fer , pH 7.4, containing 3 percent f a t t y -ac id - f ree bovine albumin, f ract ion V, in the presence and absence of the test compounds. The incubation was conducted at 37°C for 1 hour in a Dubnoff shaker osc i l l a t i ng at 40 cycles per min under an atmosphere of 95 percent 02 and 5 percent CO 2.

At the end of the incubation, the t issue was homogenized in the incu- bation medium. The concentration of free f a t t y acid in an al iquot of th is homogenate was measured by Dole's extract ion procedure (3) and Dun- combe's color imetr ic method (4). Oleic acid (Sigma) was used as the standard in the f a t t y acid extract ion and assay.

The concentration of glycerol in another al iquot of the homogenate was determined by a coupled glycerokinase-~-glycerophosphate dehydrogenase reaction (5) af ter aspirat ing the f loa t ing l i p i d layer formed by cen t r i - fugation at 1,000 x G. The amount of NADH produced was measured f luoro- me t r i ca l l y . Cer t i f ied A.C.S. grade glycerol (Fisher Sc ien t i f i c ) was used as the standard.

Results

Elevation of cyc l ic AMP. In adipose t issue of normal mice, LY79771, the R,S isomer, and LY79730, the R,R isomer, increased the concentration of cyc l ic AMP in a s ign i f i can t dose-related fashion, and the two compounds

Vol. 32, No. 13, 1983 LY79771 Stimulation of cAMP and Lipolysis 1517

were equipotent (F ig . 2). LYI03672, the S,R isomer, and LYI03085, the S,S isomer, showed no s i g n i f i c a n t dose response.

In adipose t i ssue of obese AV_Y/amice, on ly LY79771 and LY79730 at 10 -6 M s i g n i f i c a n t l y elevated c y c l i c AMP concentrat ion (Table I ) . LYI03672 and LYI03085 at the same concentrat ion had no e f f e c t .

400 -

ii UO

0

350-

300-

250-

200-

150-

100-

50-

BASAL -a -7 6 5 -4 6 -5 4

79771 79730 log [D rug ]

FIG. 2

103672 103085

Elevat ion of c y c l i c AMP in epididymal adipose t i ssue of normal mice (VY/WfL-a/a) by LY79771 and isomers. Each bar represents the mean ~ S.E. of 4 samples except fo r basal, LY79771 at 10 -6 M and LY79730 at I0 -b M, N = 8. The slope, i . e . , the average rate of change, * S.E. and i t s s i g n i - f i cance leve l fo r each isomer are:

Compound Slope • S.E. p

LY79771 (R,S) 84.52 * 13.16 <0.001 LY79730 (R,R) 83.27 * 6.39 <0.001 LYI03672 (S,R) 0.30 ± 1.06 0.787 LYI03085 (S,S) 1.98 * 0.96 0.084

The slope • S.E. and p-values were based on a l i nea r regression ana lys is .

1518 LY79771 Stimulation of cA~!P and Lipolvsis Vol. 32, No. 13, 1983

TABLE I

E levat ion of Cyc l ic AMP in Epididymal Adipose Tissue of VY/WfL-AvY/a Mice by LY79771 and Isomers at lO-6M

Add i t ion pmol/lO0 m 9 t i ssue /7 min

Basal 1 1 . 4 ~ 0.9 LY79771(R,S) 32.0 • 6.2 0.011 LY79730(R,R) 31.2 • 9.8 0.013 LYlO3672(S,R) I f . 7 • 0.8 0.966 LYIO3085(S,S) l l . 3 * l .O 0.989

The data are expressed as mean ~ S.E. of 4 samples, p-values are for comparison to the basal mean and are based on an analys is of var iance.

The 6-adrenergic property of LY79771 was inves t iga ted through the use of 6-antagonis ts : (~ ) -p rop rano lo l , a 61- and 62-blocker and a teno lo l , a se lec t i ve 61 blocker (6) . Propranolol or atenolo l alone had no e f fec t on the c y c l i c AMP level but e i t he r one of them completely reversed the e leva t ion of c y c l i c AMP by LY79771 (Table I I ) .

S t imu la t ion of l i p o l y s i s . L i po l ys i s was measured by the increase of both t o t a l f ree f a t t y acid and to ta l g l yce ro l , i . e . , the accumulation of f ree f a t t y acid and g lycero l in the t i ssue and in the incubat ion medium. A l l 4 isomers st imulated l i p o l y s i s in the adipose t i ssue of normal mice in a s i g n i f i c a n t dose-related fashion (F ig. 3). LY79771 and LY79730 were equ ipotent . LYI03085 was less potent than LY79771 and LY79730 whereas LYI03672 was the least potent .

The s i g n i f i c a n t response of AvY/A adipose t issue to LY79771 (Table I I I ) and LY79730, lO -6 M, was less than tha t of adipose t i ssue from normal mice (F ig. 3). At t h i s concent ra t ion , LYI03085 and LYI03672 were not ac t i ve .

Propranolol alone s l i g h t l y depressed the basal l i p o l y t i c rate and completely ob l i t e ra ted the l i p o l y t i c e f f e c t of LY79771. Atenolo l by i t s e l f had no e f fec t on basal rate and blocked one t h i r d (a/a) to one ha l f (AVY/a) of the s t imu la t i on caused by LY79771 (Table IV).

TABLE I I

Antagonism of IO-6M LY79771-Elevated Cyc l ic AMP in Adipose Tissue of Normal ' (a/a) and Viable Yel low Obese (Avy/a) Mice

by IO-3M (*) Propranolo l (P) or IO-3M Atenolo l (A)

AvYla p ala

Basal I I . I + 0.6 (8) 9.1 • 0.3 LY79771 33.9 ~ 3.9 (7) <0.001 42.5 • 3.5 P l l . 7 • 0.3 (4) 0.984 8.6 + 0.7 LY79771 + P l l . 2 + 0.8 (4) 0.927 8.2 * 0.4 A 11.2 ± 0.4 (4) 0.984 9.8 ± 0.3 LY79771 + A I0.8 ± 0.6 (4) 0.983 9.3 { 0.8

8) 8) <0.001 4) 0.864 4) 0.840 4) 0.920 4) 0.904

The data are expressed as mean • S.E. (N) in pmol c y c l i c AMP/IO0 mg t i ssue /7 min. p-values are fo r comparison to the basal mean and are based on an analys is of var iance.

Vol. 32, No. 13, 1983 LY79771 Stimulation of c~P and Lipolysis 1519

mm L E ~o

i | I,- :::k

2.2o 1

2"00 t 1.80

1.60 t 1.40 1.20 i 1-00 i

0.80 i 0.60 i 0.40 1 0.20 ]

0.70- =~ o.6o- Q 3

: 0.50- .=.,

°-, o 0.50- a o

o 0.20" E :::L 0.10- <3

-S - 7 - 6 - 5 - a - 8 7 6 -S a

. J . - 8 7 - 6 -S 4

79771 79730 103672 log [Drug]

, 7 S -S - 4

103085

FIG. 3

St imulat ion of l i p o l y s i s in adipose t issue of normal mice (VY/WfL-a/a) by LY79771 and isomers. Data are expressed as mean • S.E. over the basal level of 4 to 5 mice. The basal rates of l i p o l y s i s in adipose t issue of the 19 mice used in these experiments were 0.62 ~ 0.04 and 0.33 ~ 0.02 ~mol/lO0 mg t issue/h (both mean • S.E.) fo r t o ta l f ree f a t t y acid and g l yce ro l , r espec t i ve l y . The slope, i . e . , the average rate of change,

S.E. and i t s s i g n i f i c a n t level fo r each isomer were based on a l i nea r regression analys is . The p-values for each isomer are <0.001. The slopes • S.E. are:

Compound Free Fat ty Acid Glycerol

LY79771(R,S) LY79730(R,R) LYIO3672(S,R) LYI03085(S,S)

0.379 ± 0.028 0.322 * 0.062 0.081 ± 0.012 0.259 + 0.017

0.137 + 0.011 0.124 i 0.017 0.048 + 0.008 0.099 :~ 0.009

1520 LY79771 Stimulation of cAMP and Lipolysis Vol. 32, No. 13, 1983

TABLE I I I

L i p o l y t i c Ef fec t In V i t r o of LY79771 and Isomers at 10 -6 M on Epididymal Adipose Tissue of Viable Yel low Obese Mice (VY/WfL-AvY/a)

Increase of Increase of Total Free Total

Compound Fat ty Acid p Glycerol

LY79771 (R,S) 0.51 ~ 0.09 <0.001 0.23 ~ 0.03 <0.001 LY79730 (R,R) 0.47 • O.lO <O.OOl 0.22 ± 0.07 <O.OOl LYlO3672(S,R) 0.03 ± 0.08 0.574 0.03 * 0.03 0.532 LY103085(S,S) -0.05 • 0.06 0.797 0.06 • 0.06 0.188

Data are expressed as mean • S.E. over the basal leve l of 5 mice (a umol/lO0 mg t i s s u e / h ) . The basal rate of l i p o l y s i s fo r the 5 AVY/a mice was 0.80 ± 0.IO and 0.40 • 0.06 umol/lOO mg t i ssue /h fo r tot-a-i--f~ee f a t t y acid and to ta l g l yce ro l , r e s p e c t i v e l y , p-values are fo r comparison to the basal mean and are based on an analys is of var iance.

Discussion

The s t imu la t i on of c y c l i c AMP and l i p o l y s i s by LY79771 and i t s isomers in adipose t i ssue of mice is s t e reospec i f i c . The R,R isomer (LY79730) and the R,S isomer (LY79771) were much more ac t i ve than the S,S isomer (LYI03085) and the S,R isomer (LYI03672). At lower concentrat ions of compounds, the s t imu la t i on of l i p o l y s i s was propor t iona l to the increase of c y c l i c AMP in adipose t i ssue of both AVY/a and a/a mice. Compounds LYIO3672 and LYIO3085 caused l i t t l e change in cyclTc--AMP and had very l i t t l e l i p o l y t i c a c t i v i t y up to 10 -6 M. In normal adipose t i ssue , the increase of c y c l i c AMP and the s t imu la t i on of l i p o l y s i s by LY79771 and LY79730 were pa ra l l e l to each other from 10 -8 M to I0 -b M. Beyond 10 -6 M, a subs tan t ia l increase of c y c l i c AMP did not e l i c i t a p a r a l l e l increase in l i p o l y t i c response. This suggests that the a c t i v a t i o n of the l i p o l y t i c process requi res on ly a small amount of c y c l i c AMP. The large amount of c y c l i c AMP generated by the high concentrat ions of the agonists had no bearing on l i p o l y s i s .

The s t imu la t i on of c y c l i c AMP and l i p o l y s i s by LY79771 was mediated by the B-adrenergic receptor since both a c t i v i t i e s were completely blocked by propranolo l in adipose t i ssue of both a/a and AVY/a mice. The data f u r t h e r ind icated tha t the e leva t ion of c y c l i c AM-P--by-LY79771 was t o t a l l y mediated by the B 1 receptor in both genotypes of mice since a teno lo l , a se l ec t i ve B1 b locker , complete ly blocked t h i s response by the t i ssue . However, the l i p o l y t i c a c t i v i t y of LY79771 was only p a r t i a l l y reversed by atenolo l in e i t he r type of mice suggest ing tha t there is an involvement of the B2 receptor . The d i f fe rence in potency among these 4 isomers studied could be due to t h e i r i n t e r a c t i o n with the B receptors. Further i n ves t i ga t i on is necessary to p inpo in t whether t h i s d i f f e rence is due to a d i f fe rence in the b ind ing a f f i n i t y of the compounds to the receptors or due to a d i f f e rence in the i n t r i n s i c a c t i v i t y of the compounds.

The lower response of the AVY/a t i ssue to LY79771 and i t s isomers observed in t h i s study as -we'll-as to other l i p o l y t i c agents reported p rev ious l y (7) suggests the presence of a defect in the B - recep to r - cyc l i c

TAB

LE

IV

An

tag

on

ism

of

10 -6

M

LY

7977

1-S

tim

ula

ted

L

ipo

lys

is

by

10 -3

M

Pro

pra

no

lol

(P)

or

10 -3

M

Ate

no

lol

(A)

in

Ep

idid

ymal

A

dip

ose

T

issu

e o

f V

YIW

fL

Mic

e

Incr

ease

o

f T

ota

l F

ree

Fa

tty

A

cid

(a

~m

ol/

lO0

mg

tis

su

e/h

)

Incr

ease

o

f T

ota

l G

lyce

rol

AV

Y/a

p

a/a

p A

VY

/a

p a/

a

LY79

771

0.73

•

0.0

8

<0.0

01

1.39

•

0.07

<0

.001

0.

43

• 0.

04

<0.0

01

0.57

±

0.05

<0

.001

P

-0.0

3

• 0

.09

0.

380

-0.2

0

+ 0

.08

0.

008

-0.I

I *

0.0

3

0.00

3 -0

.14

+

0.0

3

0.02

4 LY

7977

1 +

P 0.

04

• 0.

05

0.61

4 0.

01

• 0.

03

0.93

7 -0

.07

i

0.05

0.

203

-0.0

6

± 0.

03

0.10

7 A

0.

01

* 0

.06

0.

455

-0.I

I ±

0.0

5

0.07

0 -0

.03

±

0.0

3

0.21

6 -0

.04

•

0.02

0.

277

LY79

771

+ A

0.

30

± 0.

09

0.00

2 0.

43

± 0.

06

<0.0

01

0.21

±

0.06

<0

.001

0

.17

±

0.03

<

0.00

1

Dat

a ar

e ex

pre

ssed

as

m

ean

• S

.E.

ove

r th

e b

asal

le

vel

of

5 m

ice.

Th

e b

asal

ra

tes

of

lip

oly

sis

fo

r a

ll

the

a/a

and

AV

Y/a

mic

e us

ed

in

thes

e ex

per

imen

ts

wer

e (m

ean

i S

.E.

in

~mo

l/lO

0 m

g ti

ss

ue

/h):

AV

Y/a

a/

a

Fre

e fa

tty

ac

id

0.77

•

0.04

0.

66

* 0.

07

Gly

cero

l 0.

48

• 0

.03

0.

35

• 0

.02

p-v

alu

es

are

for

com

par

iso

n

to

the

bas

al

mea

n a

nd

are

base

d on

an

an

alys

is

of

vari

ance

in

ea

ch

exp

erim

ent.

0 Z 0 OO

..j

0 ©

L-"

0

1522 LY79771 Stimulation of c~IP and Lipolysis Vo]. 32, No. 13, ]983

AMP system. Another manifestation of the defect in the B-receptor-cycl ic AMP system in the genet ica l ly obese mice may be observed in thermogenesis of these mice. Recent data suggest that thermogenesis is mediated by B-receptors (8, 9). Evidence has been accumulating to indicate that genet ica l ly obese rodents are e f f i c i e n t in depositing d ie tary energy through conservation of thermogenesis ( lO).

References

I . W. N. SHAW, K. K. SCHMIEGEL, T. T. YEN, R. E. TOOMEY, 0. B. MEYERS and J. MILLS, Li fe Sci. 29 2091-2101 (1981).

2. J. P. DEHAYE, J. WINAND and J. CHRISTOPHE, Diabetologia 13 553-561 (1977).

3. V. P. DOLE, J. Cl in. Invest. 35 150-154 (1956). 4. W. G. DUNCOMBE, Biochem. J. 88 7-10 (1963). 5. S. S. CHERNICK, Methods in Enzymology 14 627-630 (1969). 6. A. J. COLEMAN and A. R. SOMERVILLE, Br.--J. Pharmacol. 59 83-93 (1977). 7. T. T. T. YEN, J. STEINMETZ, and G. L. WOLFF, Horm. Meta~ol. Res. 2

200-203 (1970). 8. S. L. WELLE, D. A. THOMPSON and R. G. CAMPBELL, Am. J. Physiol. 243

R379-R382 (1982). 9. L. BUKOWIECKI, L. JAHJAH and N. FOLLEA, In t . J. Obesity 6 343-350

(1982). lO. D. L. ROMSOS, Fed. Proc. 40 2524-2529 (1981).