A C T A U N I V E R S I T A T I S L O D Z I E N S I S FOLIA BIOCHIHI CA ET BIOPHYSICA 1986_____________
M a r i a B r y e a e u a k a , C a n a r y W a t a l a , Wanda L e y k o IN VITRO EFFECT OF INSULIN ON THE OSMOTIC FRAGILITY
OF HUMAN DIABETIC ERYTHROCYTES
In v i t r o e f f e ct of in su lin on the osmotic f r a g i l i t y of human d iabetic erythrocytes was inv estigated. I t was found out that small concentrations of in s u lin (10_ , ° M - 10“9 M) increase the osmotic f r a g i l i t y rapidly and a t in su lin concentrations of 10“9 M •* 10"8 M the top values o f hemolysis were observed confirming some e a r l i e r fluorescence data. INTRODUCTION _ : ' _• .. ; i I. • D u ri n g i t s a c t i o n i n s u l i n b i n d s t o t h e s p e c i f i c r e c e p -t o r s on -t h e c e l l s u r f a c e [ l ] . S i n c e t h e n , i n s u l i n r e c e p t o r s i n s e v e r a l c e l l s su c h as a d i p o c y t e s , h e p a t o c y t e s , m o n o c y te s , g r a n u -l o c y t e s , l y m p h o c y t e s , f i b r o b l a s t s w er e fo u nd an d c h a r a c t e r i z e d . I t h a s b e e n r e c e n t l y d e i r o n s t r a t e d t h a t human [ 2 - 5 ] , t u r k e y [6] and s h e e p [7 ] e r c y t h r o c y t e s h av e a l s o s p e c i f i c i n s u l i n b i n d i n g s i t e s . T hese s i t e s h a v e b e e n c h a r a c t e r i z e d [ 4 , 8] and i t h a s b e en fo u n d o u t t h a t t h e i r p r o p e r t i e s a r e s i m i l a r t o t h o s e o f t h e i n s u l i n r e c e p t o r s i n o t h e r c e l l s . The b i n d i n g o f I - l a b e l e d i n s u l i n t o e r c y t h r o c y t e s d e p e n d s on pH, t e m p e r a t u r e , t i m e and c e l l c o n c e n t r a t i o n . The e x i s t e n c e o f i n s u l i n r e c e p t o r s i n t h e e r y t h r o c y t e membrane h a s b e e n a l r e a d y p r o v e d b u t t h e i r s i g n i f i -c a n -c e i n t h o s e -c e l l s s t i l l n e e d s e l u c i d a t i o n . ^ E r y t h r o c y t e s an d e r y t h r o c y t e membranes a r e v e r y u s e f u l f o r s t u d y i n g i n s u l i n i n t e r a c t i o n w i t h t h e c e l l u l a r membrane b e c a u s e o f t h e i r a c c e s s i b i l i t y an d e a s e o f I s o l a t i o n , t h e more so as t h e r e a r e n o t s i g n i f i c a n t d i f f e r e n c e s i n t h e hormone a c t i o n on
90 H> B ryg w n ilu, C. Watala, M. I - e y k o __________
t h e e r y t h r o c y t e membrane o r on t h e membrane o f i n s u l i n t a r g e t c e l l s .
A common a c c e p t e d model o f i n s u l i n - m e m b r a n e ■ I n t e r a c t i o n i s t h e " r e c e p t o r - t r a n s d u c e r " model w h ic h was c o n f ir m e d by many i n -v e s t i g a t i o n s [ 9 , 10J . A c c o r d in g t o t h a t model i n s u l i n I n t e r a c t s w i t h t h e membrane b i n d i n g w i t h t h e s p e c i f i c membrane r e c e p t o r - w i t h o u t e n t e r i n g t h e c e l l . The " r e c e p t o r - t r a n s d u c e r " model n e e d s s t i l l t h e d e t e r m i n a t i o n o f t h e s e c o n d m e sse n g e r o f t h e hormone i n f o r m a t i o n . I n t h e c a s e o f i n s u l i n i t s a c t i o n i s t h q u g h t t o be i n d e p e n d e n t o f c y c l i c AMP p r o d u c t i o n i n t h e c o n t r a r y t o horm ones su c h a s g l u c ag o n and a d r e n a l i n e , w hi ch s t i m u l a t e a d e n y l a t e c y -c l a s e a -c t i v i t y , so t h e n a t u r e o f t h e i n f o r m a t i o n t r a n s d u c e r r e -m ai ns unknown [ l l ] . One h y p o t h e s i s p o s t u l a t e s t h a t a l t e r a t i o n s o f membrane l i p i d s t r u c t u r e a n d / o r c h a n g e s i n membrane f l u i d i t y u n d e r i n f l u e n c e o f i n s u l i n a c t i o n may p l a y a r o l e o f a h or m o n al i n f o r m a t i o n t r a n s d u c e r [ 1 2 - 1 5 ] . The e r y t h r o c y t e membrane f l u i d i t y i s a s s o c i a t e d w i t h t h e o s -m o t ic f r a g i l i t y o f t h e e r y t h r o c y t e s . O sm otic f r a g i l i t y i s c on -s i d e r e d t o be a m e a -su r e o f e r y t h r o c y t e s v i a b i l i t y d e p e n d e n t on i t s a b i l i t y t o u n d e r g o d e f o r m a t i o n d u r i n g p a s s i n g t h r o u g h b l o o d c a p i l l a r i e s . The e r y t h r o c y t e d e f o r m a b i l i t y i s d e t e r m i n e d by t h e s u r f a c e a r e a t o volume r a t i o and by t h e e l a s t i c membrane p r o p e r -t i e s w hich e n a b l e e r y t h r o c y t e s t o u n d e r g o s t r o n g d e f o r m a t i o n s w i t h o u t r u p t u r i n g o f t h e c e l l membrane. I n t h e c a s e o f t h e e r y t h r o c y t e s f rom human s u f f e r i n g fr om d i a-b e t e s t h e d e c r e a s e d d e f o r m a b i l i t y was fo u nd [ 1 6 , 1 7 ] . B ec au se t n e d i a b e t i c e r y t h r o c y t e s a r e l e s s d e f o r m a b l e t h e y may e x e r t an i n c r e a s e d p r e s s u r e on t h e b l o o d v e s s e l w a l l s l e a d i n g t o m i c r o -a n g i o p -a t h y . The a l t e r n a t i o n s o f t h e dy nam ic p r o p e r t i e s o f t h e e r y t h r o -c y t e membrane b i l a y e r i n d i a b e t e s w e re a l s o fo un d o u t . The mi-c r o v i s mi-c o s i t y o f l i p i d b i l a y e r [18] m ea sur ed by t h e h e l p o f t h e f l u o r e s c e n c e p o l a r i z a t i o n t e c h n i q u e an d v i s c o s i t y r e l a t e d t o l a -t e r a l d i f f u s i o n o f -t h e f l u o r e s c e n -t l a b e l i n t h a t b i l a y e r [19] had much g r e a t e r v a l u e i n t h e c a s e o f t h e d i a b e t i c human e r y t h r o c y t e membranes i n d i c a t i n g an i n c r e a s e d r i g i d i t y o f t h e h y d r o p h o b i c membrane r e g i o n . I n t h i s s t u d y an e x p e r i m e n t was u n d e r t a k e n t o i n v e s t i g a t e an i n / i t r o i n f l u e n c e o f i n s u l i n on t h e o s m o t i c f r a g i l i t y o f d i a b e -t i c e r y -t h r o c y -t e s .
MATERIALS AND METHODS Bloo d fr om i n d i v i d u a l s s u f f e r i n g fr o m i n s u \ i n - d e p e n d e n t d i a -b e t e s was o -b t a i n e d f r o m D i a -b e t o l o a i c a l C l i n i c o f M e d ic a l Academy o f Ł ó d i . The p a t i e n t s w ere o f b o t h s e x e s , ( 2 5 - 5 5 ) y e a r s o l d , t r e a t e d w i t h i n s u l i n . The b l o o d was t a k e n a f t e r n i g h t f a s t , w i -t h o u -t m o rnin g d o s e o f I n s u l i n , on t h e 3% sod iu m c i t r a t e . A l l t h e p a t i e n t s showed f a r a d v a n c e d d i a b e t i c c o m p l i c a t i o n s a s p o l y n e u -r o p a t h y and r e t i n o p a t h y . D i s e a s e h ad a l o n g d u r a t i o n o f more t h a n 7 y e a r s .
F e s h l y drawn b l o o d was c e n t r i f u g e d a t 3000 rpm f o r 10 m inu-t e s . A f inu-t e r r em ov in g p l a sm a a nd l e u k o c y t e l a y e r , e r y t h r o c y t e s were f u r t h e r w ashed t h r e e t i m e s by p h o s p h a t e b u f f e r e d s a l i n e (PBS: 0.9% NaCl i n 10 mM p h o s p h a t e b u f f e r , pH 7 , 4 ) .
W ash in g was h e l d a t t e m p e r a t u r e 277 K. E r y t h r o c y t e s were t h e n b r o u g h t t o h e m a t o c r i t o f 0 . 5 . H e m a t o c r i t was d e t e r m i n e d u s -i n g a m -i c r o c e n t r -i f u g e J a n e t z k -i TH 12, t i m e o f c e n t r i f u g a t i o n 300 s . I n s u l i n ( p r e p a r a t i o n o f i n s u l i n m a x i r a p i d , p r o d u c e r s " P o l -f a " ) was added i n t o e r y t h r o c y t e s u s p e n s i o n o f h e m a t o c r i t 0 . 5 an d s am p l es w er e i n c u b a t e d a t t e m p e r a t u r e 310 K f o r 15 m i n u t e s . The e r y t h r o c y t e s u s p e n s i o n o f t h e h e m a t o c r i t 0 . 5 w i t h o r w i t h o u t i n s u l i n was n e x t ad d e d i n t o NaCl s o l u t i o n s o f d i f f e r e n t s a l t c o n c e n t r a t i o n s ( 0 . 3 8 -0 .5 0 % ) i n su c h am oun ts i n o r d e r t o o b t a i n h e m a t o c r i t o f 0 . 0 0 5 . The f i n a l i n s u l i n c o n c e n t r a t i o n s i n sam-p l e s w e re 4 • 10“ 10 M, 2 • 10_® M, 2 • 10- 8 M and 2 • 10~7 M r e -s p e c t i v e l y . The s a m p l es w ere i n c u b a t e d a t t h e t e m p e r a t u r e 310 K f o r one h o u r and t h e n c e n t r i f u g e d a t 2000 rpm f o r 10 m i n u t e s . The a b s o r b a n c e o f t h e s u p e r n a t a n t s ( h e m o l y s a t e s ) was m e a su r ed a t 542 nm. A d e g r e e o f h e m o l y s i s was c a l c u l a t e d a s su m i n g t h a t an a b s o r b a n c e o b t a i n e d a f t e r i n t r o d u c i n g t h e same am ount o f e r y -t h r o c y -t e s i n -t o d i s t i l l e d w a t e r ( t o t a l h e m o l y s i s ) i s e q u a l 100%.
RESULTS AND DISCUSSION
The o b t a i n e d r e s u l t s w er e c h a r a c t e r i z e d by a g r e a t d i s p e r -s i o n o f p e r c e n t . h e m o l y -s i -s v a l u e s f o r d i f f e r e n t i n d i v i d u a l s f o r
T h e e ff e c t o f an in c re a si n g in s u li n c o n c e n tr a ti o n « o n th e p e rc e n t o f e ry th ro c y te h e m o ly si s v a lu e fo r d if fe re n t H aC l c o n c e n tr a ti o n s (t h e p e rc e n t o f h e m o ly si s in th e a b se n ce o f in s u li n w as as sum ed e q u a l 1) W pł yw v c ra a ta ja c y c h st ę ż e ń in su li n y n a p ro ce n to w a w a rt o ć ć h e m o li z y e ry tr o c y tó w •p rr y ró ż ny ch st ę ż e n ia c h H aC l (p ro c e n t h e m o li z y b ez in su li n y p rz y ję to ja k o I) In s u li n c o n c e n tr a ti o n • H aC l c o n c e n tr a ti o n ( X ) I» } 0 .4 2 0 .4 4 0 .4 6 0 .4 8 s S .D . 0 .0 1.00 < 1 .00 1 .0 0 1. 00 1. 00 -4 .0 4 . 10 “ w 1. 08 1 .0 3 1. 03 1.0 9 1.0 6 0 .0 3 2 .0 2 • io "9 1. 10 1 .1 0 1. 25 1.1 7 1. 16 0 .0 7 -2 .0 2 • IO *8 1 .0 6 1 .07 1 .1 * 1.1 4 1 .10 0 .0 5 2 .0 2 • io “ 7 0 .9 9 0 .9 8 1.05 1.02 1.02 0 .0 3 X -m ea n o f th e p e rc e n t o f e ry th ro c y te b c m o ly si s v a lu e fo r « 11 H aC l c o n c e n tr a ti o n s. S .D . -st a n d a rd d e v ia ti o n .
t h e same NaCl c o n c e n t r a t i o n s . D u ri n g t h e r e s u l t s d e s c r i p t i o n i t was assum ed t h a t t h e p e r c e n t o f h e m o l y s i s v a l u e i n t h e a b s e n c e o f i n s u l i n i s e q u a l 1 and a ch a n g e o f t h a t v a l u e c a u s e d by i n -s u l i n o f d i f f e r e n t c o n c e n t r a t i o n -s wa-s c a l c u l a t e d . I t e n a b l e d a s t a t i s t i c a l d e s c r i p t i o n o f r e s u l t s c o n c e r n i n g t h e i n s u l i n e f f e c t on t h e o s m o t i c f r a g i l i t y o f e r y t h r o c y t e s . The o b t a i n e d r e s u l t s a r e g i v e n i n t h e t a b l e 1 , a n d t h e F i g . 1 g i v e s t h e i r g r a p h ic i n -t e r p r e -t a -t i o n . F i g . 1 r e p r e s e n t s a r e l a t i v e , mean f o r a l l NaCl c o n c e n t r a t i o n s , ch a n g e o f an e r y t h r o q y t e h e m o l y s i s p e r c e n t ( * h x ) v e r s u s t h e p e r c e n t h e m o l y s i s v a l u e i n t h e a b s e n c e o f i n s u l i n (%hQ ) , t a k e n f o r e q u a l 1, i . e . %hx/%hQ v s i n s u l i n c o n c e n t r a t i o n . An i n s e r t shows a c u r v e sh a p e f o r t h e l o w e st , i n s u l i n c o n c e n t r a -t i o n s .
The e f fe c t of an increasin g in su lin concentrations on the o sao tic f r a g i l i t y in d iab e tic human ery throcy tes. The in se r t shows a curve slope fo r the
lo-west in su lin concentrations
Wpływ wzrastających stężeń insuliny na wrażliwość osmotyczną cukrzycowych e- rytrocytdw ludzkich. Rysunek wewnętrzny przedstawia nachylenie krzywej dla
94 M. Bry»Eewn k a , C. W a ta la , W. Leyko The f i g u r e i n d i c a t e s , t h a t u n d e r t h e i n f l u e n c e o f s m a l l c o n -"»10 <~9 c e n t r a t i o n s o f i n s u l i n i n t h e r a n g e o f 10 M *• 10 M t h e osmo-t i c f r a g i l i osmo-t y i n c r e a s e s r a p i d l y r e a c h i n g a maximum f o r t h e i n -m>0 « ft s u l i n c o n c e n t r a t i o n e q u a l a b o u t 10 M *■ 10 M. F o r g r e a t e r i n s u l i n c o n c e n t r a t i o n s i t s e f f e c t on t h e o s m o t i c f r a g i l i t y d e -c r e a s e s and t h e p e r c e n t h e m o l y s i s v a l u e o f e r y t h r o c y t e s t r e n d s t o w a r d s t h e v a l u e o b s e r v e d f o r t h e a b s e n c e o f i n s u l i n (%hx/ / * h 0 - 1 ) . The p l o t s o f e r y t h r o c y t e o s m o t i c f r a g i l i t y c u r v e s w e re a l s o made w i t h o u t i n s u l i n and f o r t h a t i n s u l i n c o n c e n t r a t i o n when t h e h i g h e s t e f f e c t o f hormone on t h e i n v e s t i g a t e d v a l u e was o b s e r v e d ( 2 ‘ 10~9 M). C u rv e s a r e g i v e n i n F i g . 2 . The f i g u r e sh o w s , t h a t u n d e r t h e i n s u l i n a c t i o n t h e o s m o t ic f r a g i l i t y c u r v e i s p a r a l l e - l l y s h i f t e d t o t h e r i g h t , t o w a r d s i n c r e a s i n g NaCl c o n c e n t r a -t i o n s . The c 5Q v a l u e s f ou n d from t h e p l o t s (NaC l c o n c e n t r a -t i o n s f o r 50% o f h e m o l y s i s ) a r e e q u a l 0.436% a n d 0.443% w i t h o u t and w i t h i n s u l i n r e s p e c t i v e l y . The o b t a i n e d r e s u l t s i n d i c a t e , t h a t i n s u l i n a c t i o n d ep e n d s on horm one c o n c e n t r a t i o n an d t h e h i g h e s t e f f e c t i s o b t a i n e d f o r i n -~9 *- fl s u l i n c o n c e n t r a t i o n s i n t h e r a n g e 10 M - 10 M. The s i m i l a r s a t u r a t i o n phenomenon d u r i n g i n s u l i n a c t i o n was o b s e r v e d by L u- 1 y e t a l . [ l 3 } , who i n v e s t i g a t e d t h e i n s u l i n i n f l u e n c e on t h e r a t h e p a t o c y t e membrane f l u i d i t y and by B r y s z e w s k a e t a l . t 2©] when t h e hormone e f f e c t on t h e human e r y t h r o c y t e mem- b r a n # f l u i d i t y was i n v e s t i g a t e d . I n b o t h c a s e s t h e g r e a t e s t e f -f e c t o -f membrane v i s c o s i t y c h a n g e s was O b t a i n e d f o r i n s u l i n c o n -— ft * " — 8 c e n t r a t i o n s e q u a l 10 M and 5 • 10 M r e s p e c t i v e l y . The l a r g e s t ch a n g e o f e r y t h r o c y t e h e m o l y s i s p e r c e n t o b s e r v e d f o r i n s u l i n c o n c e n t r a t i o n 2 • 10 9 M was e q u a l a b o u t 15%. The r e - ' s u i t s o b t a i n e d p r o m p t ed u s t o s u p p o s e , t h a t i n s u l i n b i n d i n g w i t h i t s S p e c i f i c r e c e p t o r on e r y t h r o c y t e membrane c a u s e s r e m a r k a b l e c h a n g e s i n membrane s t r u c t u r e r e s u l t i n g i n an i n c r e a s e o f osmo-t i c f r a g i l i osmo-t y . The o s m o t i c f r a g i l i t y i s m a i n l y a f u n c t i o n o f t h e i n i t i a l e r y t h r o c y t e volume t o t h e c r i t i c a l volum e r a t i o v j / vc [ 2 1 ] . Thus t h e i n c r e a s i n g o s m o t ic f r a g i l i t y o f human e r y t h r o c y -t e s i n f l u e n c e d by i n s u l i n i n d i c a t e s t h a t t h e c r i t i c a l e r y t h r o c y -t e volume r e l a t e d t o h e m o l y s i s d i m i n i s h e s . I n f l u e n c e d by i n s u l i n t h e membrane becomes l e s s r e s i s t a n t t o an y d e f o r m a t i o n s and b u r s t s more r a p i d l y * The d i m i n i s h e d membrane a b i l i t y f o r d e f o r
-F ig . 2
The osmotic f r a g i l i t y curves of diabetic huui.ni erythrocytes under; the in -fluence of in sulin
Krzywe wrażliwości ostnotycznej erytrocytów ludzi chorych na cukrzycę pod wpływem insuliny
96 M. Brystewska, C. Watala, W. Leyko m a t io n s h o u l d be ac co m p an i ed by I n c r e a s e d i n s u l i n - i n d u c e d r i g i -d i t y . However i n v e s t i g a t i o n s o f l a t e r a l i r o b i l i t y ch an g es o f t h e f l u o r e s c e n t l a b e l i n t h e d i a b e t i c e r y t h r o c y t e membranes [2 0] and r o t a t i o n i r o b i l i t y c h a n g e s o f t h a t i n r a t a d i p o c y t e s membrane {12] i n d i c a t e an i n c r e a s e d f l u i d i t y o f t h e h y d r o p h o b i c membrane r e g i o n i n f l u e n c e d by i n s u l i n . On t h e o t h e r h a n d , e m p l o y in g p o l a -r i z a t i o n f l u o -r e s c e n c e t e c h n i q u e , i n c r e a s e d v i s c o s i t y r e l a t e d t o r o t a t i o n a l d i f f u s i o n o f f l u o r e s c e n t p r o b e c a u s e d by i n s u l i n i n r a t h e p a t o c y t e s was o b s e r v e d by o t h e r s [ l 3 ] , a s we^.1 a s r i g l - d i t o r y e f f e c t s o f t h e hormone t o human e r y t h r o c y t e membrane [14] w er e r e p o r t e d . M oreover some unam b igues d a t a e x i s t [ 9 , 15] wh ere no i n s u l i n - i n f l u e n c e d c h a n g es o f membrane f l u i d i t y w er e o b s e r v e d . T h e se o b s e r v a t i o n s s u g g e s t t h a t t h e i n s u l i n - i n f l u e n c e d a u gm e nt a-t i o n o f e r y t h r o c y t e o s m o t ic f r a g i l i t y d o es n o t s i m p l y c o r r e l a t e s w i t h h y d r o p h o b i c r e g i o n membrane f l u i d i t y and more l i k e l y i t i s a r e s u l t o f t h e p r o t e i n - l i p i d i n t e r a c t i o n s c h a n g es i n t h e mem-b r a n e l e a d i n g t o t h e i n c r e a s e o f e r y t h r o c y t e s e n s i b i l i t y t o h e -m o l y s i s . T h i s s t u d y was don e i n t h e c o - o p e r a t i o n w i t h t h e B i o l o g i c a l I s o t o p e L a b o r a t o r y " A . J . " o f t h e U n i v e r s i t y o f S zege d ( H un g ar y ) a nd was s u p p o r t e d by t h e R e s e a r c h G r a n t R . I I I . 1 3 . REFERENCES [ ' ] C u a t r e c a s a s P ., 1969, Proc. Natl. -457.
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Zakład Biofizyki In sty tu t Biochemii i Biofizyki UŁ
M aria B r ys ze w sk a , C ezary W a ta ia , Wanda L eyko
WPŁYW INSULINY IN VITRO NA WRAŻLIWOŚĆ 0SM0TYCZNĄ ERYTROCYTÓW LUDZI CHORYCH NA CUKRZYCg
W pracy zbadano wpływ insu lin y na wrażliwość osmotyczną erytrocytów lu-dzi chorych na cukrzyc« zależn« od insuliny. Stwierdzono, że in sul ina w
ma-łych stężeniach i.lO” , 0 M - 10~9 M) powoduje duży wzrost wrażliwości
osno--9 - 8
tycznej erytrocytów. Dla stężeń insu liny równych 10 M - 10 M obserwo-wano najwięksi« hemolitę erytrocytów, co potwierdziło u«y»k«no wcześniej wy-n ik i z pomiarów fluorescewy-ncyjwy-nych, dotyczących wpływu tego hormowy-nu wy-na ciek- łość blon erytrocytów.
