Glycogenolytic activity of Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) in vivo and in vitro

Life Sciences, Vol. 55, No. 15, pp. 1219-1228, 1994

Copyright©1994ElsevierScienceLid

Pergamon

Printed in the USA. All rights reserved 0024-3205/94 $6.00 + .00

0024-3205(94)00255.X

G L Y C O G E N O L Y T I C A C T I V I T Y OF P I T U I T A R Y A D E N Y L A T E C Y C L A S E A C T I V A T I N G P O L Y P E P T I D E (PACAP) I N V I V O A N D I N V I T R O

Y o s h i o S e k i g u c h i , K i k u o Kasai a, K a o r u H a s e g a w a b, Y o s h i n o b u Suzuki and S h i n - I c h i S h i m o d a D e p a r t m e n t of E n d o c r i n o l o g y , I n t e r n a l M e d i c i n e and b D e p a r t m e n t of P h y s i o l o g y , D o k k y o U n i v e r s i t y School of M e d i c i n e , Mibu, T o c h i g i 321-02, J a p a n

(Received in final form August 1, 1994) Summary

When pituitary adenylate cyclase activating polypeptide (PACAP38, 420 pmol/kg) was injected to anesthetized dogs, hyperglycemia associated with elevation in plasma glucagon and adrenalin levels was observed. In dogs undergone adrenalectomy, the same dose of it stimulated insulin, but not glueagon, release and hyperglycemia was not observed. Much higher dose was needed to evoke hyperglycemia in such dogs. Next, direct glycogenolytic activities of PACAP, glucagon and adrenalin were studied on cultured rat hepatocytes. PACAP38 stimulated glucose output vla cAMP production by the cells, but the potency was far less than that of glucagon. Adrenalin, however, could not exert the activity with physiological concentrations. These results indicate that hyperglycemic effect of PACAP can be attributed mainly to indirect effects resulting from glucagon and adrenalin release and possibly in part to a direct action on hepatocytes. Key Words: pituitary adenylate cyclase activation polypeptide, hyperglycemiceffect, glycogenolysis,hepatocytes

Pituitary a d e n y l a t e c y c l a s e a c t i v a t i n g p o l y p e p t i d e (PACAP) e x i s t s i n two m o l e c u l a r weight forms w i t h 38 amino a c i d r e s i d u e s (PACAP38) and w i t h 27 amino acid residues (PACAP27, which c o r r e s p o n d s to N - t e r m i n a l 27 amino a c i d r e s i dues of PACAP38) ( 1 , 2 ) . They a r e o r i g i n a l l y i s o l a t e d from t h e o v i n e hypothalami which s t i m u l a t e cAMP p r o d u c t i o n in c u l t u r e d r a t p i t u i t a r y c e l l s ( 1 , 2 ) . Based on the s t r u c t u r a l f e a t u r e s , PACAP (PACAP38 and PACAP27) b e l o n g s to a f a m i l y of peptides, which c o n s i s t of v a s o a c t i v e i n t e s t i n a l p o l y p e p t i d e (VIP), g l u c a g o n , s e c r e t i n , growth hormone r e l e a s i n g hormone (GHRH) and p e p t i d e h i s t i d i n e i s o l e u c i n e (PHI) ( 1 ) . Numerous b i o l o g i c a l a c t i v i t i e s of PACAP have been r e p o r t e d such as the s t i m u l a t i o n of the s e c r e t i o n of GH, p r o l a c t i n , ACTH and LH from the superfused pituitary c e l l s ( 1 ) , c a t e c h o l a m i n e from a d r e n o m e d u l l a r y c e l l s in yltro and i n v i v o ( 3 , 4 ) , p a n c r e a t i c amylase ( 5 ) , insulin (6), endotheliumindependent vasodilation ( 7 , 8 ) and i n o t r o p i c and c h r o n o t r o p i c a c t i o n on the heart (4). In the p r e s e n t s t u d y , e f f e c t of PACAP on g l y c o g e n o l y s i s was e v a l u ated in v i y o and i n v l t r o . aTo whom a l l c o r r e s p o n d e n c e s h o u l d be a d d r e s s e d .

1220

Glycogenolytic Action of PACAP

Materials

Vol. 55, No. 15, 1994

and Methods

I n v l v o s t u d y : F o r t y - t w o a d u l t m o n g r e l d o g s w i t h e i t h e r s e x , w e i g h i n g 7 - 10 kg w e r e u s e d f o r t h e e x p e r i m e n t . The d o g s w e r e s t a r v e d o v e r n i g h t . In o n e s t u d y , the dogs were anesthetized with sodium pentobarbiturate (26 mg/kg) i n t r a v e n o u s ly and maintained with mechanical ventilation as reported elsewhere (4,9). Following 20 - 30 min o f e q u i l i b r a t i o n , a b o l u s d o s e o f PACAP38 (270 p m o l / k g or 420 p m o l / k g ) o r r I P (420 p m o l / k g ) was a d m i n i s t e r e d intravenously at 0 time (n = 4 , 6 o r 5 ) . As c o n t r o l s (n = 5 ) , p h y s i o l o g i c a l saline was a d m i n i s t e r e d instead o f PACAP38. In another study, the dogs were anesthetized, ventilated and then bilateral adrenal glands were excised surgically. F o l l o w i n g 20 30 min e q u i l i b r a t i o n w i t h a d r i p i n f u s i o n o f 200 ml o f p h y s i o l o g i c a l saline, a b o l u s d o s e o f PACAP38 (420 p m o l / k g o r 1000 p m o l / k g ) o r a b o l u s d o s e o f PACAP38 (420 p m o l / k g ) f o l l o w e d by a n i n f u s i o n o f i t (30 p m o l / k g / m i n f o r 60 min) was administered at 0 t i m e (n = 6, 5 o r 5 ) . As c o n t r o l s (n = 6 ) , physiological s a l i n e was a d m i n i s t e r e d i n s t e a d o f PACAP38. V e n o u s w h o l e b l o o d was d r a w n a t i n d i c a t e d t i m e s , c o l l e c t e d into plastic tubes containing 5 mg o f EDTA-2Na a n d a p r o t i n i n (500 U / m l ) and immediately cooled in ice-water. P l a s m a was o b t a i n e d by c e n t r i f u g a t i o n a n d was s t o r e d a t 20 ° C u n t i l a s s a y . In vitro study: Hepatocytes were isolated f r o m m a l e W i s t a r r a t s w e i g h i n g 200 300 g a s r e p o r t e d e l s e w h e r e (10) a n d c u l t u r e d a s f o l l o w s . Hepatocytes were innoculated and cultured i n 6 w e l l p l a t e i n 2 . 0 ml o f W i l l i a m ' s E (WE) medium supplemented w i t h 2 mg/ml g l u c o s e , 50 n g / m l i n s u l i n , 1 ng/ml epidermal growth f a c t o r (EGF), 10 -6 m o l / 1 d e x a m e t h a s o n e , 5 % c a l f s e r u m a n d 2 ~ g / m l f i b r o n e c t i n at the concentration o f a r o u n d 106 c e l l s / w e l l . The medium was r e p l a c e d every day w i t h f r e s h medium f o r two d a y s . On t h e 3 r d o r 4 t h d a y , hepatocytes were gently washed three times with glucose-free Hank's balanced salt solution (GFHBSS), then preincubated i n 1 ml o f GFHBSS c o n t a i n i n g 1 mM t h e o p h y l l i n and finally i n c u b a t e d i n 1 ml o f GFHBSS w i t h 1 mM t h e o p h y l l i n e and various concentrations of a g e n t a t 37°C f o r i n d i c a t e d t i m e s . The r e a c t i o n was s t o p p e d by transferring t h e p l a t e on i c e . The medium was i m m e d i a t e l y h a r v e s t e d a n d 1 ml of 6 % ice-cold trichloroacetic a c i d (TCA) was a d d e d t o t h e c e l l s . A s s a y f o r g l u c o s e , h o r m o n e s a n d cAMP: G l u c o s e l e v e l s i n medium a n d p l a s m a w e r e m e a s u r e d by t h e k i t u s i n g m u r o t a s e - g l u c o s e o x i d a s e m e t h o d ( N a k a r a i Kagaku C o . , Tokyo, J a p a n ) . P l a s m a g l u c a g o n was m e a s u r e d by a r a d i o i m m u n o a s s a y k i t p u r c h a s e d from D a i i c h i RI L b s . ( T o k y o , J a p a n ) . The u s e d a n t i b o d y t o g l u c a g o n i n t h e kit was OAL-123 and considered to be specific to pancreatic glucagon (II). Plasma insulin was measured by a radioimmunoassay kit supplied from Eiken Kagaku (Tokyo, Japan). Plasma adrenalin levels were measured by high performance liquid chromatography in Mitsubishi Yuka Bioclinical Lab. Co.(Tokyo, Japan). Cyclic AMP levels in both medium and plasma were directly assayed and cellular cAMP levels were measured as described previously (12) by a radioimmunoassay kit supplied from Yamasa Syoyu Co. (Chiba, Japan). Chemicals: PACAP38, PACAP27, p a r t i a l polypeptides correspond to amino acid sequences o f PACAP38 f r o m 16 t o 38 (PACAP16-38) a n d f r o m 31 t o 38 (PACAP3138), vasoactive intestinal polypeptide (VIP) and glucagon were purchased from Peptide Institute Inc. (Osaka, Japan). Adrenalin bitartarate s a l t was o b t a i n e d from S i g m a (ST. L o u i s , MO, USA). Statistical statistical of variance

analysis: All data were represented a s mean ± SE o r mean ± SD, and a n a l y s i s was p e r f o r m e d by p a i r e d - t - t e s t f o l l o w i n g o n e way a n a l y s i s (ANOVA).

Vol. 55, No. 15, 1994

Glycogenolytic Action of PACAP

1221

Results As shown in Table I, plasma glucose levels were promptly increased 5 min after 420 pmol/kg PACAP38 injection and significantly higher up to 90 min with the maximum increase at 15 min when compared with the basal level. Plasma adrenalin and glueagon, but not insulin, levels Were also significantly increased after PACAP38 injection. However, in control dogs injected with physiological saline, none of these values were significantly changed. Plasma cAMP levels were promptly increased by PACAP38 injection and were significantly higher than the basal one from 5 to 30 min.

TABLE I Effects of Administration o f PACAP38(420 p m o l / k g , I V ) on P l a s m a L e v e l s o f Glucose, Adrenalin, Insulin, G l u c a g o n a n d cAMP i n A n e s t h e t i z e d Dogs. Time

Glucose

(min)

(mg/dl)

(pg/ml)

(DU/ml)

(pg/ml)

87.0± 5.7 86.6± 4.0 144.4±13.6.* 155.8±13.8.* 146.6±11.1.* 140.0± 7.5** 131.2± 6.6** 114.8± 8.5* 100.4± 7.7

9.0± 0.5 9.0± 0.5 43.0±16.5 70.0±13.0. 35.0±18.5 23.0±12.5 18.0± 7.5 i0.0± 0.5 i0.5± 0.5

7.6±2.5 9.3f2.9 9.7±2.3 9.1±2.1 7.9±2.8 8.2±2.6 8.9±2.8 9.3±2.3 7.6±1.6

88.3± 5.6 101.7±14.1 133.3±27.I* 148.6±28.4. 137.3±28.1 141.7±44.4 131.5±30.0 167.7±62.1 182.0±68.2

-15 0 5 Ib 30 45 60 90 120

The d a t a value at

Adrenalin

are represented 0 min.

Insulin

a s mean ± SE ( n : 6 ) .

Z00-

•

Glucagon

*p<0.05

8.2±0.6 9.8±0.6 20.2il.7. 22.5±5.5* 21.8±3.6. 17.3±3.6. 16.1±5.7 16.0±4.1 15.6±3.1 vs

each

PACAP38 4Z0pmoWkg iv PACAP38 2 7 0 p m o l / k g VIP 420pmok/kg tv

180

and **p<0.01

cAMP

(pmollml)

tv

160-

140-

~IzoI00-

~

80

60 -2

6

fo

4'0

6'o

8'o

;~o

i~o

TJme(min) FIG. 1 Effects levels

of a bolus injection (mean ± SE). *p<0.05

of PACAP38 and and **p<0.01VS

VIP on plasma glucose each value at O time.

Fig. 1 shows t h e h y p e r g l y c e m i c e f f e c t s o f two d o s e s of PACAP38 (270 p m o l / k g a n d 420 p m o l / k g ) a n d VIP (420 p m o l / k g ) . B o t h d o s e s o f PACAP38 c l e a r l y exhibited hyperglycemic effect. The h i g h e r d o s e o f PACAP38 (420 p m o l / k g ) h a d a greater potency t h a n t h a t o f low d o s e (270 p m o l / k g ) . VIP (420 p m o l / k g ) also exhibited hyperglycemic effect a t 5 min a f t e r i n j e c t i o n , but its effect was weaker than e v e n t h a t o f low d o s e o f PACAP38.

PG

i

i

i

0

100

200

300

400

SO0

b

30 60 time(rain)

90

FIG.

120

j

2

10

20

30

40

J

30

i

60

0

b b b °

b.c

PACAP38 iv

I0

ib

30 60 time(rain)

Ib

b

90

90

i

120

b~

120

PACAP38 420 pmol/kg iv PACAP38 1000 pmol/kg iv PACAP38 iv+DIV

control

I PACAP38 30pmol/kg/min I

0

a

I PACAP38 30pmol/kg/minJ PACAP38 iv

Effects of a bolus injection of PACAP38 (420 pmol/kg or i000 pmol/kg) and a bolus injection of PACAP38 (420 pmol/kg) followed by a continuous infusion of the agent (30 pmol/kg/min for 60 min) on plasma glucose (PG), insulin (IRI), glucagon (IRG) and cAMP levels in adrenalectomized dogs. ap<0.05 and bp<0.01 ~ each basal level at 0 time.

0

b

IPACAP38 30pmol/kg/min ] PACAP38 iv

(p! Iml)

600

(pmol/ml)

120

cAMP

90

IRG

60

0

,

30

0

L

20

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0

40

100

100[

IRI (~Ulml)

6O

IPACAP38 30pmollkglmin] PACAP38 iv

Adrenalectomy

IS0

Z00

?SO

(mgldl)

Z .o

O

<

R" >

O

Glycogenolytic Action of PACAP

Vol. 55, No. 15, 1994

1223

Next, e f f e c t s of various doses of PACAP38 on plasma glucose, insulin, glucagon and cAMPlevels were studied in b i l a t e r a l l y adrenalectomized dogs (Fig. 2). A bolus injection of PACAP38 (420 pmol/kg or i000 pmol/kg) did not exhibit hyperglycemice f f e c t . A bolus injection of PACAP38 (420 pmol/kg) f o l lowed by a continuous infusion of i t (30 pmol/kg/min for 60 m i n ) , however, c l e a r l y exhibited hyperglycemic e f f e c t , associated with a significant increase in plasma glucagon l e v e l s . In contrast to the results in dogs without adrena]ectomy, plasma insulin levels were s i g n i f i c a n t l y increased by either regimen of PACAP38 in adrenalectomized dogs. Plasma cAMP levels w e r e increased by three doses of the agent. Using primary culture of rat hepatocytes, effects of PACAP38, PACAP27, two partial polypeptides of PACAP38 (PACAPI6-38 and PACAP31-38), VIP, glucagon and adrenalin on glucose release as well as cAMP production were studied in vitro. Fig. 3 shows the time-course changes of both cAMP production and glucose output by hepatocytes incubated with 10 -6 mol/l PACAP 38. Cyclic AMP production by the cells was promptly stimulated by PACAP38 and reached the maximum level at 30 min. Release of glucose into medium from the stimulated cells by PACAP38 was linearly increased up to 15 min and thereafter gradually increased to 30 min. In contrast, cAMP production as well as glucose release by unstimulated cells was increased in a lesser extent for up to 30 min.

8

- - - e - - Control --<>-- PACAP38

60

Control

T

c)

~m

I

o

.q -6 E CL

50

Cell

40

= 20 Cl

(J

Medium

o! 0

T 10

10 0

20 min 3'0

FIG.

z

0

10

i

20

i

rain

30

3

Time-course changes of cAMP production and glucose cytes incubated with or without I0 -s mol/l PACAP38. representative of three individual wells (mean±SD).

release by hepatoThe data are

Fig. 4 shows e f f e c t s of both PACAP38 and PACAP27 on cAMP production and glucose release by hepatocytes. Both compounds dose-dependently stimulated cAMP production from 10-8 mol/1 to i0 -s mol/1, associated with a p a r a l l e l increase in glucose release, though the r e l a t i v e potency of PACAP38 appeared s l i g h t l y greater than that of PACAP27.

1224

Glycogenolytic Action of PACAP

Vol. 55, No. 15, 1994

50

c-

/Z

O 40 T ~ u

%

30

O

E

I

I0-

o. v r~

I

:E ,<

0

(.;

-9

-8

PACAP38

-7

-6

c

Log(mol/I)

Z

-9 -8 -7 PACAP38 Log(mol/I)

-6

50 [ ] Medium

O c~

[]

Cell

**

T

~" 40-

~J

% "Z o

20

-6

8

E

c'~

5" ,< (.;

C

-9

-8

PACAP27

-7

o

-6

C

-9

PACAP27

Log(tool/I)

-8

-7

-6

Log(tool/l)

FIG. 4 Effects of various doses of PACAP38 and PACAP27 on both cAMP production and glucose release by hepatocytes for 30 min. The data are represented as mean ± SD of 3 individual wells. * p<0.05 and **p<0.01 when compared with each basal value.

Two C-terminal no effect on both shown).

peptides of PACAP38 cAMP production and

such as PACAPI6-38 and PACAP31-38 glucose output by the cells (data

had not

Table II shows both cAMP production and glucose release by hepatocytes incubated with various concentrations of PACAP38 or VIP. PACAP38 dose-dependently from 10 -8 mol/l to i0 -6 mol/l stimulated glucose output as well as cAMP production by the cells. VIP also dose-dependently stimulated from 10 -7 mol/l to 10 -6 mol/l, but the potency of stimulation by it was lesser than that of PACAP38. Fig. 5 shows the effects of glucagon and adrenalin on both cAMP production and glucose output by hepatoeytes. Glucagon dose-dependently stimulated glucose output as well as cAMP production from 10 -I° mol/l to i0 -8 mol/l. The potency of 10 -6 mol/l PACAP38 was lesser than that of 10 -9 mol/l glucagon. On the other hand, adrenalin could not stimulate both cAMP production and glucose output by the cells even with 10 -8 mol/l, but much higher concentrations of it (10 -5 mol/l) clearly stimulated them.

V o l . 55, N o . 15, 1994

Glycogenolytic A c t i o n o f P A C A P

1225

TABLE I I E f f e c t s o f PACAP38 and VIP on cAMP P r o d u c t i o n tured Hepatocytes PACAP38 cAMPa log(mol/l) c e i l s medium 4.80±0.15 6.30±0.99 6.79±0.30** 8.78±0.14.* 10.75±1.35.*

Basal -

9 8 7 6

VIP cAMPa cells medium

Glucose b

0.91±0.05 1.66±0.44. 3.77±0.28** 6.22±0.36** 8.99±0.64**

and G l u c o s e R e l e a s e

4.80±0.15 4.82±0.22 5.12±0.21 6.84±0.48** 8.45±0.49**

19.6±1.8 21.6±1.5 25.2±0.9** 35.3±0.6** 37.4±1.9.*

by C u l -

Glucose b

0.91±0.05 1.10±0.13 2.01±0.45. 3.97±0.09.* 6.40±0.62**

19.6±1.8 19.5±1.3 23.0±1.9 30.9±3.2** 34.1±0.7.*

a; pmol/106 c e l l s or /ml/30 min and b; fig/106 c e l l s / 3 0 min (Mean ± SD of 3 individual w e l l s ) . *p<0.05 and **p<0.01 vs each basal l e v e l .

r-~ 100-

40[] Medium Cell

0

t

-T

z

80

"-.

O {'th

u 0

30 ~

6O

"Z

7-

20

0

-~

4o u~ o 10-

20 r~

5

o

C

-12

-II

-I0

Glucagon

70

T=

o

Ln ~W O O

60

[]

Medium

[]

Cell

-9

O_

C

12

Log(mol/l)

-11

Glucagon

'E

50

-10

-9

PACAP-6

Log(mol/I)

40-

. .

* *

30

O

m

40" 3020-

E Q.

0

PACAP-6

0

10

100 c

.

.

-12

.

-11

.

Adrenalin

.

-10

-9

-8

O

-s

c

Log(mol/I)

-lz

-11

-lo

Adrenalin

-9

-8

-5

Log(tool/I)

FIG. 5 Effects of various c o n c e n t r a t i o n s of glucagon and adrenalin on glucose r e l e a s e as well as cAMP production by hepatocytes. The data are represented as mean ± SD of 3 individual w e l l s . *p<0.05 and **p<0.01 vs each basal value.

1226

Glycogenolytic Action of PACAP

Vol. 55, No. 15, 1994

Discussion

This is the f i r s t report that PACAP exerts a hyperglycemic effect in vivo. Plasma glucose level was promptly increased a f t e r PACAP38 administration in anesthetized dogs, associated with increase in plasma levels of both adrenalin and glucagon as well as cAMP. It has been already reported that PACAP stimulates the secretion of various hormones such as GH, prolactin, ACTH and LH from superfused p i t u i t a r y c e l l s (1), catecholamines from adrenomedullary c e l l s in v i t r o and in vivo (3,4), and insulin from pancreatic i s l e t s (6). Although adrenalin secretion by PACAP in vivo has been already reported by us (4), the present study clearly showed glucagon, but not insulin, release by PACAP admini s t r a t i o n in dogs without adrenalectomy. Both adrenalin and glucagon are known to h a v e important roles on glucose metabolism. The direct e f f e c t of adrenalin on glucose delivery and clearance are mainly mediated by beta-adrenaergic mechanism and indirect e f f e c t s resulting from the inhibited secretion of insulin are mediated by alpha-adrenergic mechanisms (13). Both hormones stimulate glycogenolysis via cAMP production in l i v e r and release glucose into circulation (13,14). Accordingly, the hyperglycemia observed in vivo can be attributed to actions by both hormones released and to a possible glycogenolytic action by PACAP38 on l i v e r . PACAP belongs to a family peptides, which consist of VIP and glucagon, etc ( i ) . Especially, N-terminal amino acid sequences of PACAP38 have 68 % identity with those of VIP (1) and PACAPshares the same receptors with VIP, w h i c h have been designated as type II binding s i t e s for PACAP (15). Type II receptors are predominantly distributed on lung, l i v e r and cultured splenocytes (15). On the other hand, Type I receptors for PACAP, which are specific for PACAP but not for VIP, are predominantly distributed on adrenal gland, epididymis, anterior p i t u i t a r y , hypothalamus and cultured astrocytes (15). VIP has been already shown to exert a hyperglycemic effect fn vivo and to induce glycogenolysis in liver slices and isolated liver cells via cAMP-dependent manner (16,17). Thus, we first compared the potency of hyperglycemic effect of PACAP and VIP in vi~. The potency of PACAP38 was clearly greater than that of VIP. Accordingly, it is reasonable to consider that more potent hyperglycemic effect of PACAP than VIP may be mediated not only by its direct action on liver but also by indirect actions resulting from secretion of both adrenalin and glucagon. Thus, to eliminate an influence by adrenalin secretion after PACAP administration, we studied the effects of PACAP38 on plasma levels of glucose, insulin and glueagon in adrenalectomized dogs. In such dogs, hyperglycemic effect was not clearly observed by a bolus injection of PACAP38 (420 pmol/kg and i000 pmol/kg). Both doses of PACAP38 clearly increased plasma insulin, but not glucagon levels. Adrenalin and noradrenalin elicit beta-receptor-mediated stimulation of glucagon secretion from alpha cells, and in the presence of glucagon, alpha-2-receptor-mediated inhibition of insulin in vitro (13,14). A role for the sympathoadrenal system in regulation of endocrine pancreas, inhibition of insulin and enhancement of glucagon release, has been suggested in vivo. Accordingly, in dogs without adrenalectomy, increased adrenalin levels in plasma probably suppressed insulin secretion and inversely enhanced glucagon secretion after a bolus injection of PACAP38 (420 pmol/kg), resulting in an increase of plasma glucose levels. In contrast, in adrenalectomized dogs, a higher dose (I000 pmol/kg) as well as low dose (420 pmol/kg) of the agent could not stimulate glucagon release, but inversely stimulated insulin release which probably masked or suppressed direct glycogenolytic effect of PACAP38 on liver. Much higher dose of PACAP38(420 pmol/kg/belus and 30 pmel/kg/min for 60 min infusion) was needed to induce hyperglycemia, associated with increase in plasma levels of glucagon as well as insulin.

and

Next, we studied and compared direct effect on glycogenolysis of PACAP VIP as well as adrenalin and glucagon using rat hepatocytes in a primary

Vol. 55, No. 15, 1994

Glycogenolytic Action of PACAP

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culture. In such system, the e f f e c t of PACAP on l i v e r can be independently evaluated on glucagon and adrenalin. The in v l t r o data c l e a r l y showed t h a t both PACAP38 and PACAP27 (I0 -s - 10-6 mol/1) as well as VIP (i07- - 10-8 mol/1) dose-dependently stimulated the release of glucose, associated with an increase in cAMP production by hepatocytes. Although the potency of PACAP38 was s l i g h t ly g r e a t e r than t h a t of PACAP27 and g r e a t e r than t h a t of VIP, i t was f a r l e s s e r than t h a t of glucagon. Physiological concentrations of glucagon ( around I0 -1° mol/l) could show the a c t i v i t y , but adrenalin could not with such concentrat i o n s (10 -1° - 10-9 mol/l) in the present study. Although we did not perform the binding study for PACAP and VIP on cultured r a t hepatocytes and could not measure plasma concentration of PACAP38 a f t e r administration in vivo, the fact t h a t PACAP as well as VIP stimulated cAMP production and glucose release by hepatocytes, indicates t h a t a c t i v a t i o n of adenylate cyclase subsequent to binding to predominant Type II receptors are important to exert a glycogenolytic action. However, PACAP38 has been reported to have a higher a f f i n i t y than PACAP27 to Type I receptors, which are s p e c i f i c for PACAP and e x i s t in r a t l i v e r membranes(15,18). An action of PACAP38 through Type I receptors thus should also be considered. In c o n t r a s t to PACAP38 and PACAP27, two synthetic C-terminal peptides of PACAP38 (PACAPI6-38 and PACAP31-38) had no e f f e c t on glucose release as well as cAMP production, suggesting t h a t N-terminal sequences of PACAP38 are required for biological a c t i v i t y .

From t h e p r e s e n t i n v l ~ and i n v l t r o s t u d i e s , h y p e r g l y c e m i c e f f e c t of PACAP38 o b s e r v e d i n v i v o can be a t t r i b u t e d mainly t o i n d i r e c t a c t i o n s r e s u l t i n g from s t i m u l a t e d a d r e n a l i n and g l u c a g o n r e l e a s e and p o s s i b l y in p a r t to a d i r e c t a c t i o n on h e p a t i c g l y c o g e n o l y s i s .

Acknowledgement The authors thank to Prof. S. Yamaoka in Department of Physiology, University, School of Medicine for encouragement and useful discussion the present study.

Dokkyo during

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