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Partial purification of an insulin-releasing activity in human serum
Life Sciences, Vol. 33, pp. 2311-2319 Printed in the U.S.A.
PARTIAL
PURIFICATION
Mikael
Pergamon Press
OF AN I N S U L I N - R E L E A S I N G SERUM
ACTIVITY
IN HUMAN
Knip, 1 P e n t t i Lau~ala, 1, Hans K. Akerb~om 1 ' 2 , Kauko
Kouvalainen
and J u l i o
M. M a r t i n ~
of P e d i a t r i c s , ~Department U n i v e r s i t y of Oulu, SF-90220 O u l u 22, Children's Hospital, University of H e l s i n k i , SF-00290 H e l s i n k i 29, Finland, and R e s e a r c h Institute, H o s p i t a l for Sick Children, Toronto, Ontario, C a n a d a M5G IX8
(Received in final form September 19, 1983) SUMMARY An a c t i v i t y that e n h a n c e s insulin release from perifused rat p a n c r e a t i c islets has r e c e n t l y been isolated from human serum f r a c t i o n s (molecular weight 1,0005,000 daltons). To c h a r a c t e r i z e this a c t i v i t y we have studied the i n s u l i n - r e l e a s i n g effect of serum subfractions from obese and n o n - o b e s e children obtained by reversed-phase high-performance liquid chromatography (HPLC). The serum i n s u l i n - r e l e a s i n g a c t i v i t y eluted in the H P L C s y s t e m at 12-13 minutes, which c o r r e s p o n d e d to the r e t e n t i o n time of the t r i d e c a p e p t i d e i n s u l i n - g l u c a gon l i b e r i n isolated from bovine h y p o t h a l a m u s . Insull i n - r e l e a s i n g a c t i v i t y was found in serum s u b f r a c t i o n s from both obese and n o r m a l - w e i g h t children. The relative i n s u l i n - r e l e a s i n g p o t e n c y of the active subfractions was higher than that of the o r i g i n a l total serum fractions, indicating the p r e s e n c e of some substance(s) which inhibit insulin secretion in the total serum fractions. Oral g l u c o s e l o a d i n g increased the r e l a t i v e insulin-releasing activity in the H P L C subfractions from obese children. This study suggests that the insulin s e c r e t a g o g u e in human serum might be identical to h y p o t h a l a m i c i n s u l i n - g l u c a g o n liberin as these substances behave s i m i l a r l y on r e v e r s e d - p h a s e HPLC and have p a r a l l e l i n s u l i n - r e l e a s i n g properties.
H u m o r a l factors such as m e t a b o l i c s u b s t r a t e s as well as system i c a l l y and l o c a l l y r e l e a s e d h o r m o n e s are major r e g u l a t o r s of the insulin secretion. The e x i s t e n c e of a r e g u l a t o r y axis b e t w e e n the h y p o t h a l a m u s and the e n d o c r i n e p a n c r e a s has been d e m o n s t r a t e d in a v a r i e t y of studies (1-4). This h y p o t h a l a m o - i n s u l a r axis seems to involve both neural (5-8) and h u m o r a l p a t h w a y s (9-12). Recently, B r o u w e r et al. reported the e x t r a c t i o n from bovine h y p o t h a l a m u s of a tridecapeptide, insulin-glucagon l i b e r i n that e n h a n c e s insulin and g l u c a g o n s e c r e t i o n from p e r i f u s e d rat p a n c r e a t i c islets (13). In a p r e v i o u s study we found an i n s u l i n - r e l e a s i n g a c t i v i t y (IRA) in 0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.
2312
Serum Insulin-releasing Activity
Vol. 33, No. 23, 1983
serum fractions containing material with a molecular weight (m.wt.) of 1 , 0 0 0 - 5 , 0 0 0 d a l t o n s from obese and n o n - o b e s e c h i l d r e n (Lautala et al., s u b m i t t e d for p u b l i c a t i o n ) . To p u r i f y this serum IRA we have s u b f r a c t i o n a t e d serum f r a c t i o n s by r e v e r s e d - p h a s e highperformance liquid c h r o m a t o g r a p h y (HPLC) and found a similar sep a r a t i o n on H P L C of the serum a c t i v i t y and h y p o t h a l a m i c insuling l u c a g o n liberin, which s u g g e s t s the identity of these two substances. MATERIAL
AND METHODS
Subjects We studied six obese and six n o r m a l - w e i g h t c h i l d r e n aged 6 to 12 years. I n f o r m e d c o n s e n t was o b t a i n e d from the subjects and their parents. The relative w e i g h t of the c h i l d r e n was assessed on the basis of s t a n d a r d h e i g h t and w e i g h t charts for F i n n i s h children. O b e s i t y was d e f i n e d as a relative weight of 2 SD or more above the m e a n w e i g h t for the height. An oral glucose t o l e r a n c e test (OGTT) was p e r f o r m e d on the obese c h i l d r e n by a d m i n i s t r a t i o n of 1.75 g g l u c o s e per kg body w e i g h t (maximally i00 g glucose) after an o v e r n i g h t fast. B l o o d g l u c o s e c o n c e n t r a t i o n s were m e a s u r e d by a h e x o k i n a s e method, while the c o n c e n t r a t i o n s of serum immunoreactive insulin (IRI) were a n a l y z e d by the m e t h o d of H e r b e r t et al. (14). The s e n s i t i v i t y of the assay was 1 pU/ml. F r o m the normalweight c h i l d r e n a blood sample was o b t a i n e d after fasting for 12 hours. T a b l e I p r e s e n t s c l i n i c a l and l a b o r a t o r y data on the children studied. TABLE
I
C l i n i c a l and l a b o r a t o r y data, m e a n (SEM), n o r m a l - w e i g h t c h i l d r e n studied. O b e s e vs. • * p<0.001.
Age, yrs
Obese
children
Normal-weight
Relative weight, SD-units
in the six obese and six normal-weight: * p<0.01,
Fasting blood glucose mmol/l
Fasting
IRI
~U/ml
10.0
(1.0)
4.8
(0.6)
4.6
(0.1)
18
(4)
10.2
(I.0)
0.0
(0.2)
4.6
(0.2)
9
(i)
Peptides I n s u l i n - g l u c a g o n l i b e r i n was isolated from bovine h y p o t h a l a m u s and p u r i f i e d once by r e v e r s e d - p h a s e H P L C as p r e v i o u s l y d e s c r i b e d (13). S u b s t a n c e P, c h o l e c y s t o k i n i n (CCK) t e t r a p e p t i d e 30-33, bombesin and human ~ - e n d o r p h i n were o b t a i n e d from P e n i n s u l a L a b o r a t o ries Inc. (San Carlos, C a l i f o r n i a , USA). P o r c i n e g a s t r i c inhibitory p o p l y p e p t i d e (GIP) was p u r c h a s e d from Q u a d r a L o g i c T e c h n o l o g i e s (Victoria, B r i t i s h C o l u m b i a , Canada), while porcine g l u c a g o n was a gift from N o v o R e s e a r c h I n s t i t u t e (Bagsvaerd, Denmark).
Vol. 33, No. 23, 1983
Fractionation
of s e r u m
Serum Insulin-releasing Activity
2313
samples
To d e t e r m i n e the s e r u m IRA, blood s p e c i m e n s of 15-20 ml w e r e taken from all c h i l d r e n in a f a s t i n g state and also from obese children at 60 m i n u t e s d u r i n g the OGTT. S e r a w e r e s e p a r a t e d after clot r e t r a c t i o n and t h e r e a f t e r t r e a t e d w i t h urea, 0.643 g/ml serum, to a r r e s t the p o s s i b l e b i n d i n g and d e g r a d a t i o n of a c t i v e s u b s t a n c ~ f The samples were then successively filtrated through Amicon- " (Amicon, Danvers, Ma, USA) m e m b r a n e s XM-50, UM-10, YM-5 and UM-2 with m o l e c u l a r w e i g h t limits of 50,000, i0,000, 5,000 and 1,000 daltons, respectively. T h e m a t e r i a l r e t a i n e d by the UM-2 m e m b r a n e (m.wt. 1 , 0 0 0 - 5 , 0 0 0 daltons) was w a s h e d w i t h 0.9 % s a l i n e until the c o n c e n t r a t i o n of urea was less than 5 mmol/l. This serum fraction was s t o r e d at - 2 0 ° C at a v o l u m e of 3-6 ml until used. The conc e n t r a t i o n s of both IRI (14) and i m m u n o r e a c t i v e g l u c a g o n (IRG) (15) w e r e d e t e r m i n e d in the s e r u m fractions. For b i o a s s a y of IRA in the o r i g i n a l total s e r u m f r a c t i o n s , 0.5 ml of each f r a c t i o n was used. The r e m a i n d e r of each s e r u m f r a c t i o n was s u b j e c t e d to r e v e r s e d p h a s e HPLC. The s e p a r a t i o n s were p e r f o r m e d on a S p h e r i s o r b $ 5 0 D S 2 c o l u m n (250x4.5 mm; P h a s e Sep, Q u e e n s f e r r y , G r e a t Britain) using a V a r i a n M o d e l 8500 L i q u i d C h r o m a t o g r a p h (Varian, P a l o Alto, Ca, USA) with two s o l v e n t d e l i v e r y s y s t e m s c o u p l e d to a v a r i a b l e w a v e l e n g t h Varichrom spectrophotometer. T h e l y o p h i l i z e d p e p t i d e s t a n d a r d s and s e r u m f r a c t i o n s w e r e d i s s o l v e d for c h r o m a t o g r a p h y in the p r i m a r y solvent (0.i M N a H ~ P O 4, pH 3.10) and e l u t e d over 30 m i n u t e s at + 3 7 ° c R at a flow r ~ t e - o f 1.0 m l / m i n using a c e t o n i t r i l e (LiChrosolv ~ ), E. Merck, D a r m s t a d t , FRG) as the s e c o n d a r y s o l v e n t and a linear g r a d i e n t of 2 % a c e t o n i t r i l e / m i n . The e f f l u e n t was m o n i t o red by UV a b s o r b a n c e at 220 nm and c o l l e c t e d at one m i n u t e intervals. For r e m o v a l of the acid p h o s p h a t e buffer prior to the bioassay, the r e v e r s e d - p h a s e H P L C s u b f r a c t i o n s w e r e r e c h r o m a t o g r a p h e d on a T S K 2000 SW c o l u m n (300x7.5 mm; T o y o S o d a M a n u f a c t u r i n g Co., Tokyo, Japan) over 25 m i n u t e s at +22°C and with a flow rate of 0.5 m l / m i n using 0.9 % s a l i n e as the solvent. The m o n i t o r i n g of the e f f l u e n t was p e r f o r m e d by UV a b s o r b a n c e at 210 nm. One m i n u t e fractions were collected. O n the T S K 2000 S W c o l u m n all p e p t i d e s s t u d i e d e l u t e d b e t w e e n 19 and 21 m i n u t e s , as the r e s o l u t i o n of this steric e x c l u s i o n c o l u m n is poor in the m o l e c u l a r w e i g h t range 5005,000 d a l t o n s (16). T h e s e f r a c t i o n s were s t o r e d for s u b s e q u e n t a n a l y s i s of the IRA.
Bioassay
of the i n s u l i n - r e l e a s i n g
activity
(IRA)
The IRA of h y p o t h a l a m i c i n s u l i n - g l u c a g o n l i b e r i n m a t e r i a l , the total s e r u m f r a c t i o n s and the H P L C s u b f r a c t i o n s w e r e a s s a y e d by a m o d i f i c a t i o n of the p e r i f u s i o n t e c h n i q u e of p a n c r e a t i c islets des c r i b e d by L a c y et al. (17). T w o p a r a l l e l p e r i f u s i o n lines were used. Fifty freshly isolated ~star rat islets were p l a c e d in each tissue c h a m b e r ( M i l l i p o r e '~'' , B e d f o r d , Ma, USA). A multic h a n n e l p e r i s t a l t i c pump, l o c a t e d d i s t a l l y to the tissue c h a m b e r s , e n s u r e d a c o n s t a n t flow rate of 1.0 ml/min. The basic p e r i f u s i o n m e d i u m was K r e b s - R i n g e r b i c a r b o n a t e buffer with 0.2 % b o v i n e s e r u m albumin, 2 m M C a C l 2 and 5.5 m M glucose. It was m a i n t a i n e d at a pH of 7.4 by c o n t i n u o u s g a s s i n g with 5 % C O 2 - 9 5 % 02 . A threew a y s t o p c o c k i m m e d i a t e l y b e f o r e each t i s s u e c h a m b e r s e r v e d to admit either p e r i f u s i o n buffer or test s a m p l e w i t h a m i n i m u m of dead space. In each e x p e r i m e n t b a s a l i n s u l i n r e l e a s e was d e t e r m i n e d d u r i n g 15 m i n u t e s f o l l o w i n g an e q u i l i b r i u m p e r i o d of one hour. T h e n 0.5 ml of the s e r u m f r a c t i o n or of the H P L C s u b f r a c t i o n to be tested
2314
Serum Insulin-releasing Activity
Vol. 33, No. 23, 1983
was a d m i t t e d (Fig. 2). P e r i f u s i o n w i t h basal m e d i u m was c o n t i n u e d for 20 m i n u t e s , w h e n 0.5 ml 0.9 % s a l i n e was i n t r o d u c e d in the tissue chamber as a control. E l e v e n m i n u t e s later the islets were s t i m u l a t e d with 0.5 ml (i0 mg) g l u c o s e to assess the v i a b i l i t y of the islets. The p e r i f u s i o n was c o n t i n u e d for further 20 minutes. The insulin response to s t i m u l a t i o n was invariably monophasic, which is in a c c o r d a n c e with the t r a n s i e n t e x p o s u r e for less than one m i n u t e of the p a n c r e a t i c islets to the a c t i v e s u b s t a n c e s . Per i f u s a t e s w e r e c o l l e c t e d c o n t i n u o u s l y at one m i n u t e i n t e r v a l s and IRI was m e a s u r e d (14) at i n t e r v a l s as i n d i c a t e d in Fig. 2. Inc r e a s e s of i n s u l i n r e l e a s e w h i c h e x c e e d e d by a m i n i m u m of 2 SD the basal i n s u l i n r e l e a s e in each e x p e r i m e n t were c o n s i d e r e d as significant s t i m u l a t i o n s and the r e s p e c t i v e total s e r u m f r a c t i o n or H P L C s u b f r a c t i o n r e g a r d e d as p o s i t i v e . R e l a t i v e IRA was a s s e s s e d by relating the i n t e g r a t e d a r e a of i n s u l i n r e l e a s e above the b a s e l i n e in r e s p o n s e to the test m a t e r i a l to the i n t e g r a t e d insulin r e s p o n s e p r o v o k e d by m a x i m a l g l u c o s e s t i m u l a t i o n and e x p r e s s e d per ml of o r i g i n a l s e r u m volume. R e p e a t e d t e s t i n g (n = 5) of the r e l a t i v e IRA in two blind s e r u m f r a c t i o n s r e s u l t e d in a c o e f f i c i e n t of v a r i a t i o n of 20.4 %. T h e d a t a w e r e s t a t i s t i c a l l y e v a l u a t e d by the M a n n W h i t n e y U - t e s t and the W i l c o x o n m a t c h e d pairs signed ranks test.
RESULTS The obese c h i l d r e n had s i g n i f i c a n t l y higher s e r u m c o n c e n t r a tions of IRI in the f a s t i n g state than the n o r m a l - w e i g h t c h i l d r e n (Table I). All o b e s e c h i l d r e n had blood g l u c o s e c o n c e n t r a t i o n s w i t h i n the n o r m a l range on OGTT. The m e a n peak blood 9 1 u c o s e conc e n t r a t i o n was 8 . 0 + 0 . 4 (SEM) m m o l / l (range 6 . 0 - 8 . 9 mmol/l). The m e a n peak IRI c o n c e n t r a t i o n was 150±60 M U / m l (range 70-390 NU/ml). The o r i g i n a l total s e r u m f r a c t i o n s c o n t a i n e d no or low conc e n t r a t i o n s of IRI (range 0-3.5 N U / m l ) . The c o n c e n t r a t i o n s of IRG in the s e r u m f r a c t i o n s v a r i e d from 0 to 55 pg/ml. O n l y one of the o r i g i n a l total s e r u m f r a c t i o n s from the n o r m a l - w e i g h t c h i l d r e n was p o s i t i v e for IRA. T h r e e o b e s e c h i l d r e n had IRA in their fasting s e r u m f r a c t i o n s , w h e r e a s five w e r e p o s i t i v e for IRA at 60 m i n u t e s d u r i n g the OGTT. The r e v e r s e d - p h a s e H P L C of the i n s u l i n - g l u c a g o n l i b e r i n material showed that the p e p t i d e was not a b s o l u t e l y pure (Fig. I.A.). The HPLC subfractions corresponding to the a b s o r b a n c e peak areas were pooled, if n e c e s s a r y , and t e s t e d for b i o l o g i c a l activity. A b o u t 91 % of the total IRA was r e c o v e r e d from the h i g h e s t peak e l u t i n g at 12.6 m i n u t e s . R o u g h l y e s t i m a t e d from the o p t i c density, that peak a c c o u n t e d for m o r e than 50 % of the o r i g i n a l m a t e r i a l and a m o u n t e d to a b o u t 5 0 0 - 1 , 0 0 0 ng peptide. The other p e p t i d e s t a n d a r d s t e s t e d (substance P, C C K 30-33, GIP, b o m b e s i n , g l u c a g o n and~-endorphin) had r e t e n t i o n times w h i c h v a r i e d from 19.6 to 22.5 minutes. Fig. I.B. p r e s e n t s the c h r o m a t o g r a m of a s e r u m f r a c t i o n from an o b e s e boy on the r e v e r s e d - p h a s e column. W h e n all 30 one m i n u t e s u b f r a c t i o n s w e r e t e s t e d for IRA only f r a c t i o n 13 c o n t a i n e d a s i g n i f i c a n t IRA. A l l r e v e r s e d - p h a s e H P L C s u b f r a c t i o n s from one n o r m a l - w e i g h t c h i l d w e r e also a s s a y e d for b i o l o g i c a l a c t i v i t y and a s i g n i f i c a n t IRA was r e c o v e r e d from s u b f r a c t i o n 13. T h e r e f o r e , in the s u b s e q u e n t s t u d y o n l y the s u b f r a c t i o n s e l u t i n g at 12-13 m i n u t e s w e r e t e s t e d for IRA. T h e result of the b i o a s s a y of 1/4 (~125-250 ng p e p t i d e on the basis of the o p t i c density) of the h y p o t h a l a m i c i n s u l i n - g l u c a g o n l i b e r i n peak e l u t i n g at 12.6 m i n u t e s (Fig. I.A.) is p r e s e n t e d in Fig. 2.A. The h y p o t h a l a m i c p e p t i d e was a p o t e n t releaser of insu-
Vol. 33, No. 23, 1983
Serum Insulin-releasing Activity
2315
A.
A220nm' aufs 0.15
CH3CN, % -60
/
IGL 0.10
/
-40
/ /
0.05
- 20
0.O0
- 0
/
/
J
B.
A220nm, auf$
CH3CN, ~',
0.25
6 5 4
0.20
C3
3 II 2 0.15 - - 50
I.R A
1=Ea
t
O.10 - - 4 0
0.05 - -20
0.00 -
-o
[.~ I
0
I
5
,'0 RETENTION
I
15 TIME,
I
20
3'0
MIN
Fig. 1 R e v e r s e d - p h a s e H P L C of an i n s u l i n - r e l e a s i n g m a t e r i a l from b o v i n e h y p o t h a l a m u s (A.) p u r i f i e d once on r e v e r s e d - p h a s e H P L C as d e s c r i b e d in (13) and of a s e r u m f r a c t i o n (m.wt. 1 , 0 0 0 - 5 , 0 0 0 daltons; v o l u m e of o r i g i n a l s e r u m 4.2 ml) from an obese boy (B.). The l y o p h i l i z e d h y p o t h a l a m i c m a t e r i a l and the s e r u m f r a c t i o n w e r e d i s s o l v e d in 0.i M NaH?PO4, pH 3.10, o a n d e l u t e d on a S p h e r i s o r b $5 O D S 2 c o l u m n (250x4".5 mm) at +37 C with an a c e t o n i t r i l e gradient (2 %/min; d o t t e d line) and a flow rate of 1.0 ml/min. D e t e c t i o n by U V absorbance (220 nm). IGL = i n s u l i n - g l u c a g o n liberin; IRA = s e r u m insul i n - r e l e a s i n g activity. The n u m b e r e d boxes indicate the r e t e n t i o n time of some p e p t i d e s r e p o r t e d to s t i m u l a t e i n s u l i n release: 1 = s u b s t a n c e P, 2 = c h o l e k y s t o k i n i n t e t r a p e p t i d e 30-33, 3 = g a s t r i c inhibitory polypeptide, 4 = bombesin, 5 = pancreatic glucagon, 6 = /3 - e n d o r p h i n .
lin, the r e l a t i v e IRA b e i n g a l m o s t 2.5 times s t r o n g e r than that of 10 mg glucose. T h e m o s t a c t i v e H P L C s u b f r a c t i o n from an o b e s e ind i v i d u a l r e s u l t e d in a r e l a t i v e i n c r e a s e of the i n s u l i n r e l e a s e of 133 %/ml s e r u m (Fig. 2.B.). A l l but one of the n o r m a l - w e i g h t c h i l d r e n had IRA in the H P L C s u b f r a c t i o n , the r e l a t i v e a c t i v i t y
2316
Serum Insulin-releasing Activity
ranging from 3 to 96 %/ml serum. IRA in the H P L C s u b f r a c t i o n s than
"1
';'t
A
Vol. 33, No. 23, 1983
There was a s i g n i f i c a n t l y higher in the o r i g i n a l total serum frac-
o o;os
0 5 ML
10 MG
~ 25
}
E
20
15
,o 5ot
B
HPLC SUBFNACTION
35
1
t
zsol - c ~
oJ
,
-Is
O.S ML SALINE
10 MG GLUCOSE
t
t
2~
SASEL,NE
•
-I0
-5
0
s
10
15
~0
2s
3o
35
40
4s
50
Ss
TIME, MIN
Fig. 2. The effect of h y p o t h a l a m i c insulin-glucagon liberin (IGL; A.) purified by r e v e r s e d - p h a s e H P L C (1/4 of the peak in Fig. I . A . ; ~ 1 2 5 250 ng peptide) and of the s u b f r a c t i o n (B.) e l u t i n g at 12-13 minutes in r e v e r s e d - p h a s e H P L C of a serum fraction (original volume of the serum sample 2.8 ml) from an obese boy on the insulin release from p e r i f u s e d rat p a n c r e a t i c islets. G l u c o s e c o n c e n t r a t i o n of the p e r i f u s i o n m e d i u m was 5.5 m m o l / l and the flow rate 1.0 ml/min. The relative i n s u l i n - r e l e a s i n g a c t i v i t y (IRA) was c a l c u l a t e d by expressing the integrated area of insulin release above b a s e l i n e in response to the test s u b s t a n c e as a per cent of the insulin response e l i c i t e d by 10 mg glucose.
tions (Fig. 3). HPLC s u b f r a c t i o n s from four fasting obese c h i l d r e n were p o s i t i v e for IRA with a r e l a t i v e IRA ranging from 8 to 40 %/ml serum. All obese c h i l d r e n had b i o l o g i c a l l y active HPLC subfractions at 60 m i n u t e s during the OGTT and the relative IRA ranged from 12 to 133 %/ml serum. The relative IRA was s i g n i f i c a n t l y stronger in the H P L C s u b f r a c t i o n s than in the o r i g i n a l serum fractions at 60 m i n u t e s after the oral glucose intake. The relative IRA of the H P L C s u b f r a c t i o n s was higher in the obese c h i l d r e n after g l u c o s e loading than in the fasting state.
Vol. 33, No. 23, 1983
Serum Insulin-releasing Activity
r-p
70r - >~
2317
p < 0.05 ~ 1
60-
I-
F-
50rp
<:E
4o-
I
~
2o-
,-I
z
10-
I TOTAL FRAC -
HPLC SUB -
TOTAL FRAC -
TION
FRAC TION
TION
HPLC SUB FRAC -
TOTAL FRAC TION
TION FASTING
HPLC SUB FRAC TION
OGTT:
60 M I N
NORMAL-WEIGHT CHILDREN
OBESE
CHILDREN
Fig. 3 Relative insulin-releasing activity (IRA) in the o r i g i n a l total serum f r a c t i o n s and the s u b f r a c t i o n s eluting at 12-13 m i n u t e s on r e v e r s e d - p h a s e H P L C in six fasting n o r m a l - w e i g h t c h i l d r e n and in six obese c h i l d r e n in the fasting state and at 60 m i n u t e s after oral g l u c o s e loading. DISCUSSION This study p r o v i d e s e v i d e n c e that both n o r m a l - w e i g h t and obese c h i l d r e n have in their serum m a t e r i a l which c o e l u t e s on reversedphase H P L C with i n s u l i n - g l u c a g o n l i b e r i n isolated from bovine hypothalamus and has i n s u l i n - r e l e a s i n g p r o p e r t i e s similar to those of the h y p o t h a l a m i c t r i d e c a p e p t i d e . These observations indicate that the i n s u l i n - r e l e a s i n g m a t e r i a l in human serum may be identical to the h y p o t h a l a m i c insulin-glucagon liberin. The i n d i s t i n g u i s h a b l e c h r o m a t o g r a p h i c s e p a r a t i o n reveals similar h y d r o p h o b i c c h a r a c t e r i s tics of the two substances. The h y d r o p h o b i c p r o p e r t i e s of a peptide are on the other hand d e t e r m i n e d by the sum h y d r o p h o b i c i t y of its amino acid residues (18,19). Accordingly, the same r e t e n t i o n time on r e v e r s e d - p h a s e HPLC is indicative of c o n f o r m i t y in the m o l e c u l a r structure. The r e t e n t i o n time of the s e r u m IRA in r e v e r s e d - p h a s e H P L C excludes the p o s s i b i l i t y that the effect on insulin release could be due to known c i r c u l a t i n g p e p t i d e s with i n s u l i n - r e l e a s i n g potency, i.e. p a n c r e a t i c g l u c a g o n and GIP. S e v e r a l p e p t i d e s which are present both in the c e n t r a l n e r v o u s s y s t e m and in the g a s t r o i n t e s t i n a l tract have been found to s t i m u l a t e insulin s e c r e t i o n (20-23). Substance P appears to f a c i l i t a t e insulin r e l e a s e in vitro (20). Rehfeld et al. have isolated from nerve t e r m i n a l s in p a n c r e a t i c islets
2318
Serum Insulin-releasing Activity
Vol. 33, No. 23, 1983
the C - t e r m i n a l t e t r a p e p t i d e of c h o l e c y s t o k i n i n (CCK 30-33) and dem o n s t r a t e d that this p e p t i d e is a p o t e n t insulin releaser (21). The role of b o m b e s i n and ~ - e n d o r p h i n in the r e g u l a t i o n of insulin secretion is c o n t r o v e r s i a l (22-25). Intrapancreatically infused b o m b e s i n s t i m u l a t e s i n s u l i n s e c r e t i o n in dogs (22), but in man intr a v e n o u s i n f u s i o n of b o m b e s i n did not alter the c i r c u l a t i n g insulin c o n c e n t r a t i o n s (24). Ipp et al. found that ~ - e n d o r p h i n i n c r e a s e d insulin r e l e a s e from the p e r f u s e d c a n i n e p a n c r e a s in the p r e s e n c e of p h y s i o l o g i c a l g l u c o s e c o n c e n t r a t i o n s (23), but later the insul i n - r e l e a s i n g a c t i o n of ~ - e n d o r p h i n has been d o u b t e d (25). These four " b r a i n - g u t " p e p t i d e s can be ruled out as p o t e n t i a l c a n d i d a t e s for the s e r u m IRA on the basis of their longer r e t e n t i o n times in r e v e r s e d - p h a s e HPLC. B e l o f f - C h a i n et al. have p u r i f i e d a peptide, b e t a - c e l l tropin from the p i t u i t a r y pars i n t e r m e d i a in g e n e t i c a l l y obese mice which s t i m u l a t e s insulin release both in v i t r o and in vivo (26,27). This p e p t i d e c r o s s - r e a c t s with a C - t e r m i n a l s p e c i f i c A C T H a n t i s e r u m raised to the 17-39 s e q u e n c e of ACTH. B e t a - c e l l t r o p i n has also been isolated from the p l a s m a of g e n e t i c a l l y obese mice (28). Recently, it has been r e p o r t e d that b e t a - c e l l t r o p i n behaves s i m i l a r l y to the 22-39 fragment of A C T H on r e v e r s e d - p h a s e H P L C and that betacell t r o p i n and A C T H 22-39 have i d e n t i c a l a n t i g e n i c and i n s u l i n - r e leasing c h a r a c t e r i s t i c s (29). B a s e d on the amino acid c o m p o s i t i o n of A C T H 22-39, the r e t e n t i o n time of that p e p t i d e in our reversedp h a s e H P L C s y s t e m can be p r e d i c t e d to be about 18.5 m i n u t e s (18), i.e. c o n s i d e r a b l y longer than that of the s e r u m insulin secretagogue p u r i f i e d in the p r e s e n t study. T h e finding that the r e l a t i v e IRA was higher in the H P L C subf r a c t i o n s than in the o r i g i n a l total s e r u m fractions, suggests the p r e s e n c e of s u b s t a n c e ( s ) w h i c h inhibit insulin s e c r e t i o n in the latter. In c o n s i d e r i n g the m o l e c u l a r w e i g h t range, s o m a t o s t a t i n is a p o s s i b l e c a n d i d a t e for that effect. The study indicates that oral g l u c o s e intake p o t e n t i a t e s the s e r u m IRA at least in obese children. Hence, the s e r u m IRA m a y mod i f y the insulin r e s p o n s e to oral glucose. In a recent study we found that the p r o p o r t i o n of serum f r a c t i o n s p o s i t i v e for IRA were higher in obese c h i l d r e n than in n o r m a l - w e i g h t c h i l d r e n (Lautala, s u b m i t t e d for p u b l i c a t i o n ) . In o b e s e c h i l d r e n the r e l a t i v e IRA of the total s e r u m f r a c t i o n s c o r r e l a t e d p o s i t i v e l y with the d u r a t i o n and d e g r e e of obesity. T h e r e was also an a s s o c i a t i o n b e t w e e n IRA and g r o w t h velocity. T h o s e o b s e r v a t i o n s implied that the s e r u m IRA m a y be i n v o l v e d in the r e g u l a t i o n of body w e i g h t and linear growth. The lack of s i g n i f i c a n t d i f f e r e n c e s in the r e l a t i v e IRA b e t w e e n obese and n o n - o b e s e c h i l d r e n in the p r e s e n t study was e x p e c t e d owing to the small number of p a t i e n t s studied. The p h y s i o l o g i c a l signif i c a n c e of the insulin s e c r e t a g o g u e in h u m a n s e r u m remains to be defined. N e v e r t h e l e s s , on the basis of our findings we p o s t u l a t e that the s e r u m " i n s u l i n liberin" m a y play a role in the r e g u l a t i o n of the e n d o c r i n e p a n c r e a s as a link in the h y p o t h a l a m o - i n s u l a r axis.
ACKNOWLEDGEMENTS T h i s s t u d y was s u p p o r t e d by the M e d i c a l R e s e a r c h C o u n c i l of Finland (MK), the S i g n e and Ane G y l l e n b e r g F o u n d a t i o n ( H K A ) , the A l m a and K.A. S n e l l m a n F o u n d a t i o n , Oulu, F i n l a n d (MK) and F a r m o s G r o u p s Ltd. The S i g r i d J u s 4 1 i u s F o u n d a t i o n m a d e regular p e r s o n a l e x c h a n g e b e t w e e n O u l u and T o r o n t o p o s s i b l e . W e w i s h to thank S i r p a
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A n t t i l a and M a t t i V u o t i for their e x p e r t t e c h n i c a l a s s i s t a n c e . Our thanks are also due to A s s o c i a t e P r o f e s s o r J u h a n i L e p p ~ l u o t o , M.D., for his v a l u a b l e a d v i c e d u r i n g v a r i o u s s t a g e s of this work. REFERENCES i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
25. 26. 27. 28. 29.
L.A. F R O H M A N and L.L. B E R N A R D I S . E n d o c r i n o l o g y 82, 1 1 2 5 - 1 1 3 2 (1968). P.W. HAN, and L.A. FROHMAN. Am. J. Physiol. 219, 1 6 3 2 - 1 6 3 6 (1970). B.E. H U S T V E D T , and A. L@V0. A c t a Physiol. scand. 84, 29-33 (1972). J.M. M A R T I N , W. K O N I N J N E N D I J K , and P.R. BOUMAN. D i a b e t e s 23 203-208 (1974). T.L. POWLEY, and C.A. OPSDAL. Am. J. P h y s i o l . 226, 25-33 (1974). S. INOUE, and G.A. BRAY. E n d o c r i n o l o g y i00, 108-114 (1977). H.R. B E R T H O U D , and B. J E A N R E N A U D . E n d o c r i n o l o g y 105, 1 4 6 - 1 5 1 (1979). F. R O H N E R - J E A N R E N A U D , and B. J E A N R E N A U D . J. clin. Invest. 65, 902-910 (1980). L.A. IDAHL, and J.M. MARTIN. J. Endocrinol. 51, 601-602 (1971). R.B. L O C K H A R T - E W A R T , C. MOK, and J.M. MARTIN. D i a b e t e s 25, 96i00 (1976). J.H. MOLTZ, R.E. DOBBS, S.M. M c C A N N , and C.P. FAWCETT. Endoc r i n o l o g y 105, 1 2 6 2 - 1 2 6 8 , (1979). E. B O B B I O N I , and B. J E A N R E N A U D . Endocrinology ii0, 631-636 (1982). G.H. B R O U W E R , M.S. LAMPTEY, and J.M. MARTIN. L i f e Sci. 30, 703-710 (1982) . V. H E R B E R T , K.S. LAU, C.W. G O T T L I E B , and S.J. B L E I C H E R . J. clin. E n d o c r i n o l . 25, 1 3 7 5 - 1 3 8 4 (1965). L.G. HEDING. D i a b e t o l o g i a 7, 10-19 (1971). S. R O H U S H I K A , T. O K H A W A , and H. HATANO. J. C h r o m a t o g r . 176, 4 5 6 - 4 6 1 (1979). P.E. LACY, E.H. FINKE, S.M. CONANT, and S. NABER. D i a b e t e s 25, 4 8 4 - 4 9 1 (1976). J.L. MEEK. Proc. natl. Acad. Sci. U S A 77, 1 6 3 2 - 1 6 3 6 (1980). T. S A S A G A W A , T. O K O Y A M A , and D.C. TELLER. J. C h r o m a t o g r . 240, 329-340 (1982). K. H E R M A N S E N . E n d o c r i n o l o g y 104, 832-838 (1980). J.H. R E H F E L D , L.I. LARSSON, N.R. G O L T E R M A N , T.W. S C H W A R T Z , J.K. HOLST, S.L. J E N S E N , and J.S. MORLEY. N a t u r e 284, 33-38 (1980). A. KANETO, T. KANETO, S. NAKAYA, H. K A J I N U M A , and K. KOSAKA. M e t a b o l i s m 27, 549-553 (1978). E. IPP, R. DOBBS, and R.H. UNGER. N a t u r e 276, 1 9 0 - 1 9 1 (1978). E. LEZOCHE, N. BASSO, and V. S P E R A N Z A . In Gut H o r m o n e s , e d i t e d by S.R. BLOOM, and J.M. POLAK, pp. 419-424, C h u r c h i l l - L i v i n g stone, E d i n b u r g h (1981). G. S C H A F F E R - T A N N E N B A U M , A.E. P A N E R A I A , and H.G. F R I E S E N . Life Sci. 25, 1 9 8 3 - 1 9 9 0 (1980). A. B E L O F F - C H A I N , S. D U N M O R E , and J. M O R T O N . FEBS L e t t e r s 117, 303-307 (1980). A. B E L O F F - C H A I N , P. CARR, and A. W A T K I N S O N . FEBS L e t t e r s 139, 230-232 (1982). N. B I L L I N G H A M , A. B E L O F F - C H A I N , and M.A. C A W T H O R N E . J. Endocrinol. 94, 125-130 (1982). A° B E L O F F - C H A I N , J. M O R T O N , S. D U N M O R E , G.W. T A Y L O R , and H.R. MORRIS. N a t u r e 301, 255-258 (1983).
PARTIAL
PURIFICATION
Mikael
Pergamon Press
OF AN I N S U L I N - R E L E A S I N G SERUM
ACTIVITY
IN HUMAN
Knip, 1 P e n t t i Lau~ala, 1, Hans K. Akerb~om 1 ' 2 , Kauko
Kouvalainen
and J u l i o
M. M a r t i n ~
of P e d i a t r i c s , ~Department U n i v e r s i t y of Oulu, SF-90220 O u l u 22, Children's Hospital, University of H e l s i n k i , SF-00290 H e l s i n k i 29, Finland, and R e s e a r c h Institute, H o s p i t a l for Sick Children, Toronto, Ontario, C a n a d a M5G IX8
(Received in final form September 19, 1983) SUMMARY An a c t i v i t y that e n h a n c e s insulin release from perifused rat p a n c r e a t i c islets has r e c e n t l y been isolated from human serum f r a c t i o n s (molecular weight 1,0005,000 daltons). To c h a r a c t e r i z e this a c t i v i t y we have studied the i n s u l i n - r e l e a s i n g effect of serum subfractions from obese and n o n - o b e s e children obtained by reversed-phase high-performance liquid chromatography (HPLC). The serum i n s u l i n - r e l e a s i n g a c t i v i t y eluted in the H P L C s y s t e m at 12-13 minutes, which c o r r e s p o n d e d to the r e t e n t i o n time of the t r i d e c a p e p t i d e i n s u l i n - g l u c a gon l i b e r i n isolated from bovine h y p o t h a l a m u s . Insull i n - r e l e a s i n g a c t i v i t y was found in serum s u b f r a c t i o n s from both obese and n o r m a l - w e i g h t children. The relative i n s u l i n - r e l e a s i n g p o t e n c y of the active subfractions was higher than that of the o r i g i n a l total serum fractions, indicating the p r e s e n c e of some substance(s) which inhibit insulin secretion in the total serum fractions. Oral g l u c o s e l o a d i n g increased the r e l a t i v e insulin-releasing activity in the H P L C subfractions from obese children. This study suggests that the insulin s e c r e t a g o g u e in human serum might be identical to h y p o t h a l a m i c i n s u l i n - g l u c a g o n liberin as these substances behave s i m i l a r l y on r e v e r s e d - p h a s e HPLC and have p a r a l l e l i n s u l i n - r e l e a s i n g properties.
H u m o r a l factors such as m e t a b o l i c s u b s t r a t e s as well as system i c a l l y and l o c a l l y r e l e a s e d h o r m o n e s are major r e g u l a t o r s of the insulin secretion. The e x i s t e n c e of a r e g u l a t o r y axis b e t w e e n the h y p o t h a l a m u s and the e n d o c r i n e p a n c r e a s has been d e m o n s t r a t e d in a v a r i e t y of studies (1-4). This h y p o t h a l a m o - i n s u l a r axis seems to involve both neural (5-8) and h u m o r a l p a t h w a y s (9-12). Recently, B r o u w e r et al. reported the e x t r a c t i o n from bovine h y p o t h a l a m u s of a tridecapeptide, insulin-glucagon l i b e r i n that e n h a n c e s insulin and g l u c a g o n s e c r e t i o n from p e r i f u s e d rat p a n c r e a t i c islets (13). In a p r e v i o u s study we found an i n s u l i n - r e l e a s i n g a c t i v i t y (IRA) in 0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.
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serum fractions containing material with a molecular weight (m.wt.) of 1 , 0 0 0 - 5 , 0 0 0 d a l t o n s from obese and n o n - o b e s e c h i l d r e n (Lautala et al., s u b m i t t e d for p u b l i c a t i o n ) . To p u r i f y this serum IRA we have s u b f r a c t i o n a t e d serum f r a c t i o n s by r e v e r s e d - p h a s e highperformance liquid c h r o m a t o g r a p h y (HPLC) and found a similar sep a r a t i o n on H P L C of the serum a c t i v i t y and h y p o t h a l a m i c insuling l u c a g o n liberin, which s u g g e s t s the identity of these two substances. MATERIAL
AND METHODS
Subjects We studied six obese and six n o r m a l - w e i g h t c h i l d r e n aged 6 to 12 years. I n f o r m e d c o n s e n t was o b t a i n e d from the subjects and their parents. The relative w e i g h t of the c h i l d r e n was assessed on the basis of s t a n d a r d h e i g h t and w e i g h t charts for F i n n i s h children. O b e s i t y was d e f i n e d as a relative weight of 2 SD or more above the m e a n w e i g h t for the height. An oral glucose t o l e r a n c e test (OGTT) was p e r f o r m e d on the obese c h i l d r e n by a d m i n i s t r a t i o n of 1.75 g g l u c o s e per kg body w e i g h t (maximally i00 g glucose) after an o v e r n i g h t fast. B l o o d g l u c o s e c o n c e n t r a t i o n s were m e a s u r e d by a h e x o k i n a s e method, while the c o n c e n t r a t i o n s of serum immunoreactive insulin (IRI) were a n a l y z e d by the m e t h o d of H e r b e r t et al. (14). The s e n s i t i v i t y of the assay was 1 pU/ml. F r o m the normalweight c h i l d r e n a blood sample was o b t a i n e d after fasting for 12 hours. T a b l e I p r e s e n t s c l i n i c a l and l a b o r a t o r y data on the children studied. TABLE
I
C l i n i c a l and l a b o r a t o r y data, m e a n (SEM), n o r m a l - w e i g h t c h i l d r e n studied. O b e s e vs. • * p<0.001.
Age, yrs
Obese
children
Normal-weight
Relative weight, SD-units
in the six obese and six normal-weight: * p<0.01,
Fasting blood glucose mmol/l
Fasting
IRI
~U/ml
10.0
(1.0)
4.8
(0.6)
4.6
(0.1)
18
(4)
10.2
(I.0)
0.0
(0.2)
4.6
(0.2)
9
(i)
Peptides I n s u l i n - g l u c a g o n l i b e r i n was isolated from bovine h y p o t h a l a m u s and p u r i f i e d once by r e v e r s e d - p h a s e H P L C as p r e v i o u s l y d e s c r i b e d (13). S u b s t a n c e P, c h o l e c y s t o k i n i n (CCK) t e t r a p e p t i d e 30-33, bombesin and human ~ - e n d o r p h i n were o b t a i n e d from P e n i n s u l a L a b o r a t o ries Inc. (San Carlos, C a l i f o r n i a , USA). P o r c i n e g a s t r i c inhibitory p o p l y p e p t i d e (GIP) was p u r c h a s e d from Q u a d r a L o g i c T e c h n o l o g i e s (Victoria, B r i t i s h C o l u m b i a , Canada), while porcine g l u c a g o n was a gift from N o v o R e s e a r c h I n s t i t u t e (Bagsvaerd, Denmark).
Vol. 33, No. 23, 1983
Fractionation
of s e r u m
Serum Insulin-releasing Activity
2313
samples
To d e t e r m i n e the s e r u m IRA, blood s p e c i m e n s of 15-20 ml w e r e taken from all c h i l d r e n in a f a s t i n g state and also from obese children at 60 m i n u t e s d u r i n g the OGTT. S e r a w e r e s e p a r a t e d after clot r e t r a c t i o n and t h e r e a f t e r t r e a t e d w i t h urea, 0.643 g/ml serum, to a r r e s t the p o s s i b l e b i n d i n g and d e g r a d a t i o n of a c t i v e s u b s t a n c ~ f The samples were then successively filtrated through Amicon- " (Amicon, Danvers, Ma, USA) m e m b r a n e s XM-50, UM-10, YM-5 and UM-2 with m o l e c u l a r w e i g h t limits of 50,000, i0,000, 5,000 and 1,000 daltons, respectively. T h e m a t e r i a l r e t a i n e d by the UM-2 m e m b r a n e (m.wt. 1 , 0 0 0 - 5 , 0 0 0 daltons) was w a s h e d w i t h 0.9 % s a l i n e until the c o n c e n t r a t i o n of urea was less than 5 mmol/l. This serum fraction was s t o r e d at - 2 0 ° C at a v o l u m e of 3-6 ml until used. The conc e n t r a t i o n s of both IRI (14) and i m m u n o r e a c t i v e g l u c a g o n (IRG) (15) w e r e d e t e r m i n e d in the s e r u m fractions. For b i o a s s a y of IRA in the o r i g i n a l total s e r u m f r a c t i o n s , 0.5 ml of each f r a c t i o n was used. The r e m a i n d e r of each s e r u m f r a c t i o n was s u b j e c t e d to r e v e r s e d p h a s e HPLC. The s e p a r a t i o n s were p e r f o r m e d on a S p h e r i s o r b $ 5 0 D S 2 c o l u m n (250x4.5 mm; P h a s e Sep, Q u e e n s f e r r y , G r e a t Britain) using a V a r i a n M o d e l 8500 L i q u i d C h r o m a t o g r a p h (Varian, P a l o Alto, Ca, USA) with two s o l v e n t d e l i v e r y s y s t e m s c o u p l e d to a v a r i a b l e w a v e l e n g t h Varichrom spectrophotometer. T h e l y o p h i l i z e d p e p t i d e s t a n d a r d s and s e r u m f r a c t i o n s w e r e d i s s o l v e d for c h r o m a t o g r a p h y in the p r i m a r y solvent (0.i M N a H ~ P O 4, pH 3.10) and e l u t e d over 30 m i n u t e s at + 3 7 ° c R at a flow r ~ t e - o f 1.0 m l / m i n using a c e t o n i t r i l e (LiChrosolv ~ ), E. Merck, D a r m s t a d t , FRG) as the s e c o n d a r y s o l v e n t and a linear g r a d i e n t of 2 % a c e t o n i t r i l e / m i n . The e f f l u e n t was m o n i t o red by UV a b s o r b a n c e at 220 nm and c o l l e c t e d at one m i n u t e intervals. For r e m o v a l of the acid p h o s p h a t e buffer prior to the bioassay, the r e v e r s e d - p h a s e H P L C s u b f r a c t i o n s w e r e r e c h r o m a t o g r a p h e d on a T S K 2000 SW c o l u m n (300x7.5 mm; T o y o S o d a M a n u f a c t u r i n g Co., Tokyo, Japan) over 25 m i n u t e s at +22°C and with a flow rate of 0.5 m l / m i n using 0.9 % s a l i n e as the solvent. The m o n i t o r i n g of the e f f l u e n t was p e r f o r m e d by UV a b s o r b a n c e at 210 nm. One m i n u t e fractions were collected. O n the T S K 2000 S W c o l u m n all p e p t i d e s s t u d i e d e l u t e d b e t w e e n 19 and 21 m i n u t e s , as the r e s o l u t i o n of this steric e x c l u s i o n c o l u m n is poor in the m o l e c u l a r w e i g h t range 5005,000 d a l t o n s (16). T h e s e f r a c t i o n s were s t o r e d for s u b s e q u e n t a n a l y s i s of the IRA.
Bioassay
of the i n s u l i n - r e l e a s i n g
activity
(IRA)
The IRA of h y p o t h a l a m i c i n s u l i n - g l u c a g o n l i b e r i n m a t e r i a l , the total s e r u m f r a c t i o n s and the H P L C s u b f r a c t i o n s w e r e a s s a y e d by a m o d i f i c a t i o n of the p e r i f u s i o n t e c h n i q u e of p a n c r e a t i c islets des c r i b e d by L a c y et al. (17). T w o p a r a l l e l p e r i f u s i o n lines were used. Fifty freshly isolated ~star rat islets were p l a c e d in each tissue c h a m b e r ( M i l l i p o r e '~'' , B e d f o r d , Ma, USA). A multic h a n n e l p e r i s t a l t i c pump, l o c a t e d d i s t a l l y to the tissue c h a m b e r s , e n s u r e d a c o n s t a n t flow rate of 1.0 ml/min. The basic p e r i f u s i o n m e d i u m was K r e b s - R i n g e r b i c a r b o n a t e buffer with 0.2 % b o v i n e s e r u m albumin, 2 m M C a C l 2 and 5.5 m M glucose. It was m a i n t a i n e d at a pH of 7.4 by c o n t i n u o u s g a s s i n g with 5 % C O 2 - 9 5 % 02 . A threew a y s t o p c o c k i m m e d i a t e l y b e f o r e each t i s s u e c h a m b e r s e r v e d to admit either p e r i f u s i o n buffer or test s a m p l e w i t h a m i n i m u m of dead space. In each e x p e r i m e n t b a s a l i n s u l i n r e l e a s e was d e t e r m i n e d d u r i n g 15 m i n u t e s f o l l o w i n g an e q u i l i b r i u m p e r i o d of one hour. T h e n 0.5 ml of the s e r u m f r a c t i o n or of the H P L C s u b f r a c t i o n to be tested
2314
Serum Insulin-releasing Activity
Vol. 33, No. 23, 1983
was a d m i t t e d (Fig. 2). P e r i f u s i o n w i t h basal m e d i u m was c o n t i n u e d for 20 m i n u t e s , w h e n 0.5 ml 0.9 % s a l i n e was i n t r o d u c e d in the tissue chamber as a control. E l e v e n m i n u t e s later the islets were s t i m u l a t e d with 0.5 ml (i0 mg) g l u c o s e to assess the v i a b i l i t y of the islets. The p e r i f u s i o n was c o n t i n u e d for further 20 minutes. The insulin response to s t i m u l a t i o n was invariably monophasic, which is in a c c o r d a n c e with the t r a n s i e n t e x p o s u r e for less than one m i n u t e of the p a n c r e a t i c islets to the a c t i v e s u b s t a n c e s . Per i f u s a t e s w e r e c o l l e c t e d c o n t i n u o u s l y at one m i n u t e i n t e r v a l s and IRI was m e a s u r e d (14) at i n t e r v a l s as i n d i c a t e d in Fig. 2. Inc r e a s e s of i n s u l i n r e l e a s e w h i c h e x c e e d e d by a m i n i m u m of 2 SD the basal i n s u l i n r e l e a s e in each e x p e r i m e n t were c o n s i d e r e d as significant s t i m u l a t i o n s and the r e s p e c t i v e total s e r u m f r a c t i o n or H P L C s u b f r a c t i o n r e g a r d e d as p o s i t i v e . R e l a t i v e IRA was a s s e s s e d by relating the i n t e g r a t e d a r e a of i n s u l i n r e l e a s e above the b a s e l i n e in r e s p o n s e to the test m a t e r i a l to the i n t e g r a t e d insulin r e s p o n s e p r o v o k e d by m a x i m a l g l u c o s e s t i m u l a t i o n and e x p r e s s e d per ml of o r i g i n a l s e r u m volume. R e p e a t e d t e s t i n g (n = 5) of the r e l a t i v e IRA in two blind s e r u m f r a c t i o n s r e s u l t e d in a c o e f f i c i e n t of v a r i a t i o n of 20.4 %. T h e d a t a w e r e s t a t i s t i c a l l y e v a l u a t e d by the M a n n W h i t n e y U - t e s t and the W i l c o x o n m a t c h e d pairs signed ranks test.
RESULTS The obese c h i l d r e n had s i g n i f i c a n t l y higher s e r u m c o n c e n t r a tions of IRI in the f a s t i n g state than the n o r m a l - w e i g h t c h i l d r e n (Table I). All o b e s e c h i l d r e n had blood g l u c o s e c o n c e n t r a t i o n s w i t h i n the n o r m a l range on OGTT. The m e a n peak blood 9 1 u c o s e conc e n t r a t i o n was 8 . 0 + 0 . 4 (SEM) m m o l / l (range 6 . 0 - 8 . 9 mmol/l). The m e a n peak IRI c o n c e n t r a t i o n was 150±60 M U / m l (range 70-390 NU/ml). The o r i g i n a l total s e r u m f r a c t i o n s c o n t a i n e d no or low conc e n t r a t i o n s of IRI (range 0-3.5 N U / m l ) . The c o n c e n t r a t i o n s of IRG in the s e r u m f r a c t i o n s v a r i e d from 0 to 55 pg/ml. O n l y one of the o r i g i n a l total s e r u m f r a c t i o n s from the n o r m a l - w e i g h t c h i l d r e n was p o s i t i v e for IRA. T h r e e o b e s e c h i l d r e n had IRA in their fasting s e r u m f r a c t i o n s , w h e r e a s five w e r e p o s i t i v e for IRA at 60 m i n u t e s d u r i n g the OGTT. The r e v e r s e d - p h a s e H P L C of the i n s u l i n - g l u c a g o n l i b e r i n material showed that the p e p t i d e was not a b s o l u t e l y pure (Fig. I.A.). The HPLC subfractions corresponding to the a b s o r b a n c e peak areas were pooled, if n e c e s s a r y , and t e s t e d for b i o l o g i c a l activity. A b o u t 91 % of the total IRA was r e c o v e r e d from the h i g h e s t peak e l u t i n g at 12.6 m i n u t e s . R o u g h l y e s t i m a t e d from the o p t i c density, that peak a c c o u n t e d for m o r e than 50 % of the o r i g i n a l m a t e r i a l and a m o u n t e d to a b o u t 5 0 0 - 1 , 0 0 0 ng peptide. The other p e p t i d e s t a n d a r d s t e s t e d (substance P, C C K 30-33, GIP, b o m b e s i n , g l u c a g o n and~-endorphin) had r e t e n t i o n times w h i c h v a r i e d from 19.6 to 22.5 minutes. Fig. I.B. p r e s e n t s the c h r o m a t o g r a m of a s e r u m f r a c t i o n from an o b e s e boy on the r e v e r s e d - p h a s e column. W h e n all 30 one m i n u t e s u b f r a c t i o n s w e r e t e s t e d for IRA only f r a c t i o n 13 c o n t a i n e d a s i g n i f i c a n t IRA. A l l r e v e r s e d - p h a s e H P L C s u b f r a c t i o n s from one n o r m a l - w e i g h t c h i l d w e r e also a s s a y e d for b i o l o g i c a l a c t i v i t y and a s i g n i f i c a n t IRA was r e c o v e r e d from s u b f r a c t i o n 13. T h e r e f o r e , in the s u b s e q u e n t s t u d y o n l y the s u b f r a c t i o n s e l u t i n g at 12-13 m i n u t e s w e r e t e s t e d for IRA. T h e result of the b i o a s s a y of 1/4 (~125-250 ng p e p t i d e on the basis of the o p t i c density) of the h y p o t h a l a m i c i n s u l i n - g l u c a g o n l i b e r i n peak e l u t i n g at 12.6 m i n u t e s (Fig. I.A.) is p r e s e n t e d in Fig. 2.A. The h y p o t h a l a m i c p e p t i d e was a p o t e n t releaser of insu-
Vol. 33, No. 23, 1983
Serum Insulin-releasing Activity
2315
A.
A220nm' aufs 0.15
CH3CN, % -60
/
IGL 0.10
/
-40
/ /
0.05
- 20
0.O0
- 0
/
/
J
B.
A220nm, auf$
CH3CN, ~',
0.25
6 5 4
0.20
C3
3 II 2 0.15 - - 50
I.R A
1=Ea
t
O.10 - - 4 0
0.05 - -20
0.00 -
-o
[.~ I
0
I
5
,'0 RETENTION
I
15 TIME,
I
20
3'0
MIN
Fig. 1 R e v e r s e d - p h a s e H P L C of an i n s u l i n - r e l e a s i n g m a t e r i a l from b o v i n e h y p o t h a l a m u s (A.) p u r i f i e d once on r e v e r s e d - p h a s e H P L C as d e s c r i b e d in (13) and of a s e r u m f r a c t i o n (m.wt. 1 , 0 0 0 - 5 , 0 0 0 daltons; v o l u m e of o r i g i n a l s e r u m 4.2 ml) from an obese boy (B.). The l y o p h i l i z e d h y p o t h a l a m i c m a t e r i a l and the s e r u m f r a c t i o n w e r e d i s s o l v e d in 0.i M NaH?PO4, pH 3.10, o a n d e l u t e d on a S p h e r i s o r b $5 O D S 2 c o l u m n (250x4".5 mm) at +37 C with an a c e t o n i t r i l e gradient (2 %/min; d o t t e d line) and a flow rate of 1.0 ml/min. D e t e c t i o n by U V absorbance (220 nm). IGL = i n s u l i n - g l u c a g o n liberin; IRA = s e r u m insul i n - r e l e a s i n g activity. The n u m b e r e d boxes indicate the r e t e n t i o n time of some p e p t i d e s r e p o r t e d to s t i m u l a t e i n s u l i n release: 1 = s u b s t a n c e P, 2 = c h o l e k y s t o k i n i n t e t r a p e p t i d e 30-33, 3 = g a s t r i c inhibitory polypeptide, 4 = bombesin, 5 = pancreatic glucagon, 6 = /3 - e n d o r p h i n .
lin, the r e l a t i v e IRA b e i n g a l m o s t 2.5 times s t r o n g e r than that of 10 mg glucose. T h e m o s t a c t i v e H P L C s u b f r a c t i o n from an o b e s e ind i v i d u a l r e s u l t e d in a r e l a t i v e i n c r e a s e of the i n s u l i n r e l e a s e of 133 %/ml s e r u m (Fig. 2.B.). A l l but one of the n o r m a l - w e i g h t c h i l d r e n had IRA in the H P L C s u b f r a c t i o n , the r e l a t i v e a c t i v i t y
2316
Serum Insulin-releasing Activity
ranging from 3 to 96 %/ml serum. IRA in the H P L C s u b f r a c t i o n s than
"1
';'t
A
Vol. 33, No. 23, 1983
There was a s i g n i f i c a n t l y higher in the o r i g i n a l total serum frac-
o o;os
0 5 ML
10 MG
~ 25
}
E
20
15
,o 5ot
B
HPLC SUBFNACTION
35
1
t
zsol - c ~
oJ
,
-Is
O.S ML SALINE
10 MG GLUCOSE
t
t
2~
SASEL,NE
•
-I0
-5
0
s
10
15
~0
2s
3o
35
40
4s
50
Ss
TIME, MIN
Fig. 2. The effect of h y p o t h a l a m i c insulin-glucagon liberin (IGL; A.) purified by r e v e r s e d - p h a s e H P L C (1/4 of the peak in Fig. I . A . ; ~ 1 2 5 250 ng peptide) and of the s u b f r a c t i o n (B.) e l u t i n g at 12-13 minutes in r e v e r s e d - p h a s e H P L C of a serum fraction (original volume of the serum sample 2.8 ml) from an obese boy on the insulin release from p e r i f u s e d rat p a n c r e a t i c islets. G l u c o s e c o n c e n t r a t i o n of the p e r i f u s i o n m e d i u m was 5.5 m m o l / l and the flow rate 1.0 ml/min. The relative i n s u l i n - r e l e a s i n g a c t i v i t y (IRA) was c a l c u l a t e d by expressing the integrated area of insulin release above b a s e l i n e in response to the test s u b s t a n c e as a per cent of the insulin response e l i c i t e d by 10 mg glucose.
tions (Fig. 3). HPLC s u b f r a c t i o n s from four fasting obese c h i l d r e n were p o s i t i v e for IRA with a r e l a t i v e IRA ranging from 8 to 40 %/ml serum. All obese c h i l d r e n had b i o l o g i c a l l y active HPLC subfractions at 60 m i n u t e s during the OGTT and the relative IRA ranged from 12 to 133 %/ml serum. The relative IRA was s i g n i f i c a n t l y stronger in the H P L C s u b f r a c t i o n s than in the o r i g i n a l serum fractions at 60 m i n u t e s after the oral glucose intake. The relative IRA of the H P L C s u b f r a c t i o n s was higher in the obese c h i l d r e n after g l u c o s e loading than in the fasting state.
Vol. 33, No. 23, 1983
Serum Insulin-releasing Activity
r-p
70r - >~
2317
p < 0.05 ~ 1
60-
I-
F-
50rp
<:E
4o-
I
~
2o-
,-I
z
10-
I TOTAL FRAC -
HPLC SUB -
TOTAL FRAC -
TION
FRAC TION
TION
HPLC SUB FRAC -
TOTAL FRAC TION
TION FASTING
HPLC SUB FRAC TION
OGTT:
60 M I N
NORMAL-WEIGHT CHILDREN
OBESE
CHILDREN
Fig. 3 Relative insulin-releasing activity (IRA) in the o r i g i n a l total serum f r a c t i o n s and the s u b f r a c t i o n s eluting at 12-13 m i n u t e s on r e v e r s e d - p h a s e H P L C in six fasting n o r m a l - w e i g h t c h i l d r e n and in six obese c h i l d r e n in the fasting state and at 60 m i n u t e s after oral g l u c o s e loading. DISCUSSION This study p r o v i d e s e v i d e n c e that both n o r m a l - w e i g h t and obese c h i l d r e n have in their serum m a t e r i a l which c o e l u t e s on reversedphase H P L C with i n s u l i n - g l u c a g o n l i b e r i n isolated from bovine hypothalamus and has i n s u l i n - r e l e a s i n g p r o p e r t i e s similar to those of the h y p o t h a l a m i c t r i d e c a p e p t i d e . These observations indicate that the i n s u l i n - r e l e a s i n g m a t e r i a l in human serum may be identical to the h y p o t h a l a m i c insulin-glucagon liberin. The i n d i s t i n g u i s h a b l e c h r o m a t o g r a p h i c s e p a r a t i o n reveals similar h y d r o p h o b i c c h a r a c t e r i s tics of the two substances. The h y d r o p h o b i c p r o p e r t i e s of a peptide are on the other hand d e t e r m i n e d by the sum h y d r o p h o b i c i t y of its amino acid residues (18,19). Accordingly, the same r e t e n t i o n time on r e v e r s e d - p h a s e HPLC is indicative of c o n f o r m i t y in the m o l e c u l a r structure. The r e t e n t i o n time of the s e r u m IRA in r e v e r s e d - p h a s e H P L C excludes the p o s s i b i l i t y that the effect on insulin release could be due to known c i r c u l a t i n g p e p t i d e s with i n s u l i n - r e l e a s i n g potency, i.e. p a n c r e a t i c g l u c a g o n and GIP. S e v e r a l p e p t i d e s which are present both in the c e n t r a l n e r v o u s s y s t e m and in the g a s t r o i n t e s t i n a l tract have been found to s t i m u l a t e insulin s e c r e t i o n (20-23). Substance P appears to f a c i l i t a t e insulin r e l e a s e in vitro (20). Rehfeld et al. have isolated from nerve t e r m i n a l s in p a n c r e a t i c islets
2318
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the C - t e r m i n a l t e t r a p e p t i d e of c h o l e c y s t o k i n i n (CCK 30-33) and dem o n s t r a t e d that this p e p t i d e is a p o t e n t insulin releaser (21). The role of b o m b e s i n and ~ - e n d o r p h i n in the r e g u l a t i o n of insulin secretion is c o n t r o v e r s i a l (22-25). Intrapancreatically infused b o m b e s i n s t i m u l a t e s i n s u l i n s e c r e t i o n in dogs (22), but in man intr a v e n o u s i n f u s i o n of b o m b e s i n did not alter the c i r c u l a t i n g insulin c o n c e n t r a t i o n s (24). Ipp et al. found that ~ - e n d o r p h i n i n c r e a s e d insulin r e l e a s e from the p e r f u s e d c a n i n e p a n c r e a s in the p r e s e n c e of p h y s i o l o g i c a l g l u c o s e c o n c e n t r a t i o n s (23), but later the insul i n - r e l e a s i n g a c t i o n of ~ - e n d o r p h i n has been d o u b t e d (25). These four " b r a i n - g u t " p e p t i d e s can be ruled out as p o t e n t i a l c a n d i d a t e s for the s e r u m IRA on the basis of their longer r e t e n t i o n times in r e v e r s e d - p h a s e HPLC. B e l o f f - C h a i n et al. have p u r i f i e d a peptide, b e t a - c e l l tropin from the p i t u i t a r y pars i n t e r m e d i a in g e n e t i c a l l y obese mice which s t i m u l a t e s insulin release both in v i t r o and in vivo (26,27). This p e p t i d e c r o s s - r e a c t s with a C - t e r m i n a l s p e c i f i c A C T H a n t i s e r u m raised to the 17-39 s e q u e n c e of ACTH. B e t a - c e l l t r o p i n has also been isolated from the p l a s m a of g e n e t i c a l l y obese mice (28). Recently, it has been r e p o r t e d that b e t a - c e l l t r o p i n behaves s i m i l a r l y to the 22-39 fragment of A C T H on r e v e r s e d - p h a s e H P L C and that betacell t r o p i n and A C T H 22-39 have i d e n t i c a l a n t i g e n i c and i n s u l i n - r e leasing c h a r a c t e r i s t i c s (29). B a s e d on the amino acid c o m p o s i t i o n of A C T H 22-39, the r e t e n t i o n time of that p e p t i d e in our reversedp h a s e H P L C s y s t e m can be p r e d i c t e d to be about 18.5 m i n u t e s (18), i.e. c o n s i d e r a b l y longer than that of the s e r u m insulin secretagogue p u r i f i e d in the p r e s e n t study. T h e finding that the r e l a t i v e IRA was higher in the H P L C subf r a c t i o n s than in the o r i g i n a l total s e r u m fractions, suggests the p r e s e n c e of s u b s t a n c e ( s ) w h i c h inhibit insulin s e c r e t i o n in the latter. In c o n s i d e r i n g the m o l e c u l a r w e i g h t range, s o m a t o s t a t i n is a p o s s i b l e c a n d i d a t e for that effect. The study indicates that oral g l u c o s e intake p o t e n t i a t e s the s e r u m IRA at least in obese children. Hence, the s e r u m IRA m a y mod i f y the insulin r e s p o n s e to oral glucose. In a recent study we found that the p r o p o r t i o n of serum f r a c t i o n s p o s i t i v e for IRA were higher in obese c h i l d r e n than in n o r m a l - w e i g h t c h i l d r e n (Lautala, s u b m i t t e d for p u b l i c a t i o n ) . In o b e s e c h i l d r e n the r e l a t i v e IRA of the total s e r u m f r a c t i o n s c o r r e l a t e d p o s i t i v e l y with the d u r a t i o n and d e g r e e of obesity. T h e r e was also an a s s o c i a t i o n b e t w e e n IRA and g r o w t h velocity. T h o s e o b s e r v a t i o n s implied that the s e r u m IRA m a y be i n v o l v e d in the r e g u l a t i o n of body w e i g h t and linear growth. The lack of s i g n i f i c a n t d i f f e r e n c e s in the r e l a t i v e IRA b e t w e e n obese and n o n - o b e s e c h i l d r e n in the p r e s e n t study was e x p e c t e d owing to the small number of p a t i e n t s studied. The p h y s i o l o g i c a l signif i c a n c e of the insulin s e c r e t a g o g u e in h u m a n s e r u m remains to be defined. N e v e r t h e l e s s , on the basis of our findings we p o s t u l a t e that the s e r u m " i n s u l i n liberin" m a y play a role in the r e g u l a t i o n of the e n d o c r i n e p a n c r e a s as a link in the h y p o t h a l a m o - i n s u l a r axis.
ACKNOWLEDGEMENTS T h i s s t u d y was s u p p o r t e d by the M e d i c a l R e s e a r c h C o u n c i l of Finland (MK), the S i g n e and Ane G y l l e n b e r g F o u n d a t i o n ( H K A ) , the A l m a and K.A. S n e l l m a n F o u n d a t i o n , Oulu, F i n l a n d (MK) and F a r m o s G r o u p s Ltd. The S i g r i d J u s 4 1 i u s F o u n d a t i o n m a d e regular p e r s o n a l e x c h a n g e b e t w e e n O u l u and T o r o n t o p o s s i b l e . W e w i s h to thank S i r p a
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A n t t i l a and M a t t i V u o t i for their e x p e r t t e c h n i c a l a s s i s t a n c e . Our thanks are also due to A s s o c i a t e P r o f e s s o r J u h a n i L e p p ~ l u o t o , M.D., for his v a l u a b l e a d v i c e d u r i n g v a r i o u s s t a g e s of this work. REFERENCES i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
25. 26. 27. 28. 29.
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