UBC Theses and Dissertations

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UBC Theses and Dissertations

Hypertension and diabetic cardiomyopathy Rodrigues, Brian Baltzar 1985

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H Y P E R T E N S I O N A N D D I A B E T I C C A R D I O M Y O P A T H Y by B r i a n B a l t z a r R o d r i g u e s B . S c , K a r a c h i U n i v e r s i t y , P a k i s t a n , 1977; M . S c , K a r a c h i U n i v e r s i t y , P a k i s t a n , 1978 A T h e s i s S u b m i t t e d in P a r t i a l F u l f i l m e n t o f T h e R e q u i r e m e n t s f o r the D e g r e e of M a s t e r of S c i e n c e in T h e F a c u l t y of G r a d u a t e S t u d i e s ( D i v i s i o n of P h a r m a c o l o g y and T o x i c o l o g y in the F a c u l t y of P h a r m a c e u t i c a l Sc iences ) W e a c c e p t th is thes is as c o n f o r m i n g to the r e q u i r e d s t a n d a r d s : T h e U n i v e r s i t y of B r i t i s h C o l u m b i a S e p t e m b e r 1985 ' ^ b r i a n B a l t z a r R o d r i g u e s , 1985 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of PH¥) ft i^y f\c o L O U The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6C3/81) i i A B S T R A C T T h e i s o l a t e d p e r f u s e d w o r k i n g h e a r t was used to s t u d y h y p e r -t e n s i v e - d i a b e t e s i n d u c e d a l t e r a t i o n s i n c a r d i a c f u n c t i o n at 6 and 12 w e e k s a f t e r t h e i n d u c t i o n o f d i a b e t e s . T h e r e w a s no d i f f e r e n c e i n c a r d i a c f u n c t i o n b e t w e e n n o r m o t e n s i v e W i s t a r and s p o n t a n e o u s l y h y p e r t e n s i v e (SHR) d i a b e t i c r a t s at 6 w e e k s a f t e r d i a b e t e s i n d u c t i o n . W i s t a r - K y o t o ( W K Y ) r a t s w e r e a lso i n c l u d e d as n o r m o t e n s i v e c o n t r o l s i n our 12-week s t u d y . S u c c e s s f u l i n d u c t i o n o f d iabe tes was c o n f i r m e d by the p r e s e n c e of h y p e r g l y c e m i a , h y p o i n s u l i n e m i a , g l y c o s u r i a and i n c r e a s e d h a e m o g l o b i n g l y c o s y l a t i o n in a l l t h r e e d i a b e t i c groups . H o w e v e r , q u a n t i t a t i o n of v a r i o u s p a r a m e t e r s of hear t f u n c t i o n r e v e a l e d h i g h l y s i g n i f i c a n t d i f f e r e n c e s b e t w e e n S H R d i a b e t i c a n i m a l s and a l l o t h e r groups , a s s o c i a t e d w i t h an i n c r e a s e d m o r t a l i t y . S e r u m l i p i d s w e r e e l e v a t e d i n S H R and W i s t a r and u n a f f e c t e d i n W K Y d i a b e t i c r a t s . F u r t h e r m o r e , t h y r o i d h o r m o n e l e v e l s w e r e not d e p r e s s e d i n W K Y d i a b e t i c r a t s and c o u l d e x p l a i n t h e l a c k of c a r d i a c d y s f u n c t i o n i n these a n i m a l s . T h e d a t a p r o v i d e f u r t h e r e v i d e n c e that the c o m b i n a t i o n o f h y p e r t e n s i o n and d i a b e t e s m e l l i t u s p r o d u c e s g r e a t e r m y o c a r d i a l d y s f u n c t i o n t h a n is seen w i t h e i t h e r d isease a lone and is a s s o c i a t e d w i t h a s i g n i f i c a n t m o r t a l i t y . T h e e f f e c t s o f h y d r a l a z i n e on b l o o d l i p i d s , s y s t o l i c pressure and c a r d i a c p e r f o r m a n c e w e r e assessed i n m a l e W i s t a r r a t s , 6 w e e k s a f t e r t h e y w e r e m a d e d i a b e t i c w i t h s t r e p t o z o t o c i n (STZ) . W h e n h y d r a l a z i n e was a d m i n i s t e r e d f o r a 6 - w e e k p e r i o d to the d i a b e t i c r a t s , t h e i r b l o o d l i p i d s w e r e not s i g n i f i c a n t l y d i f f e r e n t f r o m that o f n o n - d i a b e t i c r a t s d e s p i t e a l o w s e r u m i n s u l i n . In c o n t r a s t , b l o o d l i p i d s w e r e e l e v a t e d i n the d i a b e t i c r a t s tha t w e r e not t r e a t e d w i t h h y d r a l a z i n e ; these a n i m a l s i i i a l so had l o w i n s u l i n l e v e l s . C a r d i a c p e r f o r m a n c e was depressed in the u n t r e a t e d d i a b e t i c a n i m a l s , but the c a r d i a c p e r f o r m a n c e o f the h y d r a l a z i n e - t r e a t e d d i a b e t i c a n i m a l s s h o w e d a d e f i n i t e i m p r o v e m e n t w h i c h c o u l d be p a r t l y e x p l a i n e d by t h e i r n o r m a l t h y r o i d s ta tus i n c o n t r a s t to the u n t r e a t e d d i a b e t i c a n i m a l s w h i c h w e r e s l i g h t l y h y p o t h y r o i d . B l o o d p r e s s u r e was e l e v a t e d o n l y i n the u n t r e a t e d d i a b e t i c a n i m a l s . T h u s h y d r a -l a z i n e c o n t r o l l e d the h igh s e r u m l i p i d s and b l o o d pressure and i m p r o v e d c a r d i a c p e r f o r m a n c e i n S T Z d i a b e t i c r a t s . T o e x a m i n e the i n f l u e n c e of sex d i f f e r e n c e s i n the S T Z m o d e l of d i a b e t e s , w e s t u d i e d l e f t v e n t r i c u l a r f u n c t i o n in hear t s f r o m 6 w e e k m a l e and f e m a l e r a t s . S i g n i f i c a n t l y l o w e r v a l u e s f o r +dP/dt o c c u r r e d i n m a l e d i a b e t i c r a t s c o m p a r e d w i t h t h e i r o w n c o n t r o l s or f e m a l e d i a b e t i c s at m o s t l e f t a t r i a l f i l l i n g pressures . D e c r e a s e s i n this v a l u e f o r f e m a l e d i a b e t i c r a t s c o m p a r e d to t h e i r o w n c o n t r o l s o c c u r r e d o n l y at h i g h l e f t a t r i a l pressures . It appears that d i a b e t e s m e l l i t u s p r o d u c e s g r e a t e r m y o c a r d i a l d y s f u n c t i o n i n m a l e d i a b e t i c r a t s . J o h n H . M c N e i l l , P h . D . P r o f e s s o r and D e a n F a c u l t y of P h a r m a c e u t i c a l S c i e n c e s T h e s i s S u p e r v i s o r C O N T E N T S A B S T R A C T L I S T O F T A B L E S L I S T O F F I G U R E S L I S T O F A B B R E V I A T I O N S A C K N O W L E D G E M E N T S I N T R O D U C T I O N : A . O v e r v i e w B . P a t h o p h y s i o l o g y o f D i a b e t e s C . D r u g s and C h e m i c a l s tha t p r o d u c e d i a b e t e s D . D i a b e t i c c a r d i o m y o p a t h y E . D i a b e t e s and a r t e r i a l h y p e r t e n s i o n F . H y d r a l a z i n e (an a n t i h y p e r t e n s i v e agent) G . Sex d i f f e r e n c e s i n the s t r e p t o z o t o c i n m o d e l o f d i a b e t e s S P E C I F I C G O A L S O F T H E I N V E S T I G A T I O N M A T E R I A L S A N D M E T H O D S A . M A T E R I A L S : 1. C h e m i c a l s 2. A s s a y k i t s B . M E T H O D S 1. I n d u c t i o n o f d i a b e t e s 2. T r e a t m e n t p r o t o c o l s a) S i x w e e k s S H R s tudy b) T w e l v e w e e k s S H R s t u d y c) H y d r a l a z i n e s tudy iv P A G E i i v i v i i ix x 1 6 9 10 12 14 15 17 18 18 18 18 19 19 20 P A G E d) P r e p a r a t i o n o f i s o l a t e d t issues 21 e) M a l e and f e m a l e s t u d y 21 3. W o r k i n g h e a r t p e r f u s i o n . 22 4. M e a s u r e m e n t of s y s t o l i c b l o o d pressure and h e a r t r a t e o f 24 u n a n e s t h e t i z e d r a t s 5. B l o o d analyses 25 6. S t a t i s t i c a l analyses 26 R E S U L T S 27 D I S C U S S I O N 82 C O N C L U S I O N S 105 R E F E R E N C E S 106 A B S T R A C T S 118 P A P E R S 119 L I S T O F T A B L E S 1. C l a s s i f i c a t i o n o f d i a b e t e s m e l l i t u s and r e l a t e d g lucose i n t o l e r a n c e s ( S H R study) 2. C e r t a i n f e a t u r e s o f 6 w e e k s c o n t r o l and d i a b e t i c r a t s ( S H R study) 3. V a r i o u s p a r a m e t e r s used to assess the d i a b e t i c s t a t e o f 12 w e e k d i a b e t i c a n i m a l s ( S H R study) 4. G e n e r a l c h a r a c t e r i s t i c s o f 12 w e e k s c o n t r o l and d i a b e t i c r a t s ( S H R study) 5. E f f e c t s o f s t r e p t o z o t o c i n (STZ) p r e t r e a t m e n t on s y s t o l i c b l o o d pressure (mm Hg) i n 12 w e e k s c o n t r o l and d i a b e t i c r a t s ( S H R study) 6. G e n e r a l f e a t u r e s of the e x p e r i m e n t a l r a t s ( h y d r a l a z i n e study) 7. V a r i o u s p a r a m e t e r s used to assess the d i a b e t i c s t a t e o f the e x p e r i m e n t a l r a t s ( h y d r a l a z i n e study) 8. E f f e c t s of s t r e p t o z o t o c i n (STZ) p r e t r e a t m e n t on s y s t o l i c b l o o d pressure (mm Hg) o f c o n t r o l and d i a b e t i c r a t s ( h y d r a -l a z i n e s t u d y ) . 9. V a r i o u s p a r a m e t e r s used to assess the d i a b e t i c s t a t e o f e x p e r i m e n t a l r a t s (male and f e m a l e study) v i i L I S T O F F I G U R E S P A G E 1. T h e a c u t e e f f e c t s of i n s u l i n d e p r i v a t i o n 8 2. E f f e c t s o f d i a b e t e s and h y p e r t e n s i o n on 30 (A) l e f t v e n t r i c u l a r d e v e l o p e d pressure , (B) +dP/dt and (C) - d P / d t o f i s o l a t e d p e r f u s e d w o r k i n g h e a r t s at v a r i o u s f i l l i n g pressures 3. E f f e c t s of d iabe tes on body w e i g h t of S H R , W i s t a r 34 and W K Y d i a b e t i c r a t s a f t e r the i n d u c t i o n of d i a b e t e s w i t h 55 m g / k g i . v . s t r e p t o z o t o c i n 4. E f f e c t o f d i a b e t e s on h e a r t r a t e o f S H R , W K Y and 39 W i s t a r r a t s 5. A - D - S e r u m f r e e f a t t y a c i d s , t r i g l y c e r i d e s , c h o l e s t e r o l 41 and phosphol ip ids i n S H R , W K Y and W i s t a r r a t s 12 w e e k s a f t e r s t r e p t o z o t o c i n i n j e c t i o n 6. A - C - E f f e c t s o f d i a b e t e s and h y p e r t e n s i o n on 50 (A) l e f t v e n t r i c u l a r d e v e l o p e d pressure ; (B) +dP/dt , and (C) - d P / d t o f i s o l a t e d p e r f u s e d w o r k i n g h e a r t s at v a r i o u s f i l l i n g pressures 7. E f f e c t s of S T Z - i n d u c e d d i a b e t e s (12 weeks) on 56 m y o c a r d i a l f u n c t i o n o f p e r f u s e d h e a r t s i s o l a t e d f r o m W i s t a r c o n t r o l , S H R c o n t r o l , W K Y c o n t r o l , W K Y d i a b e t i c , W i s t a r d i a b e t i c and S H R d i a b e t i c r a t s 8. E f f e c t o f d i a b e t e s and h y d r a l a z i n e t r e a t m e n t on hear t r a t e o f e x p e r i m e n t a l r a t s 60 9. E f f e c t of d i a b e t e s and h y d r a l a z i n e t r e a t m e n t on (A) l e f t v e n t r i c u l a r d e v e l o p e d p r e s s u r e , (B) - d P / d t , and (C) +dP/dt of i s o l a t e d p e r f u s e d w o r k i n g h e a r t s at v a r i o u s f i l l i n g pressures 10. E f f e c t o f d i a b e t e s and h y d r a l a z i n e t r e a t m e n t on s e r u m f r e e f a t t y ac ids of e x p e r i m e n t a l r a t s 11. E f f e c t o f d i a b e t e s and h y d r a l a z i n e t r e a t m e n t on s e r u m t r i g l y c e r i d e s o f e x p e r i m e n t a l r a t s 12. E f f e c t o f d i a b e t e s and h y d r a l a z i n e t r e a t m e n t on s e r u m t o t a l c h o l e s t e r o l of e x p e r i m e n t a l r a t s 13. E f f e c t o f d i a b e t e s and h y d r a l a z i n e t r e a t m e n t on s e r u m phosphol ip ids o f e x p e r i m e n t a l r a t s 14. D o s e - r e s p o n s e c u r v e s o f h y d r a l a z i n e on tens ion and r a t e d e v e l o p m e n t o f i s o l a t e d l e f t and r i g h t a t r i a o b t a i n e d f r o m 6 w e e k c o n t r o l and S T Z - t r e a t e d r a t s 15. E f f e c t of d i a b e t e s on +dP/dt in (A) F e m a l e and (B) M a l e i s o l a t e d p e r f u s e d w o r k i n g h e a r t s at v a r i o u s f i l l i n g pressures 16. L i p o p r o t e i n p a t h w a y s and m a j o r s teps in the r e g u l a t i o n o f t r i g l y c e r i d e m e t a b o l i s m i n the a d i p o c y t e i x L I S T O F A B B R E V I A T I O N S S H R W K Y S T Z +dP/dt - d P / d t T 4 T 3 c y c l i c A M P S p o n t a n e o u s l y h y p e r t e n s i v e ra t W i s t a r - K y o t o S t r e p t o z o t o c i n R a t e of l e f t v e n t r i c u l a r pressure d e v e l o p m e n t R a t e o f l e f t v e n t r i c u l a r pressure d e c l i n e 3 ,5 ,3 ' ,5 ' t e t r a i o d o t h y r o n i n e 3 ,5 ,3 ' t r i i o d o t h y r o n i n e C y c l i c adenosine 5 ' - m o n o - p h o s p h a t e X A C K N O W L E D G E M E N T S I w i s h to express m y deepest g r a t i t u d e to m y s u p e r v i s o r D r . John H . M c N e i l l , D e a n , whose d i r e c t i o n , i n t e r e s t and p a t i e n c e e n a b l e d m e to c o m p l e t e this w o r k . I w o u l d a lso l i k e to express s i n c e r e thanks to D r . C l a y t o n H e y l i g e r and D r . R a m e s h G o y a l f o r t h e i r genuine i n t e r e s t and e n c o u r a g e m e n t throughout the c o u r s e of the s t u d y . T h e suggest ions and s c i e n t i f i c input f r o m the m e m b e r s o f the s u p e r v i s o r y c o m m i t t e e , e s p e c i a l l y D r . C a t h y P a n g , are g r a t e f u l l y a c k n o w l e d g e d . I a m g r a t e f u l to D r . A r u n T a h i l i a n i , S h a b i r B h i m j i and B r u c e W i l s o n f o r t h e i r i n v a l u a b l e t e c h n i c a l a s s i s t a n c e . I am e s p e c i a l l y g r a t e f u l to M s . J u d y W y n e f o r t y p i n g this thes i s . B r i a n B . R o d r i g u e s XI T O M Y P A R E N T S , N I C H O L A S A N D A N N I E F O R A L L T H E Y H A V E D O N E . W I T H L O V E . 1 INTRODUCTION A. OVERVIEW Diabetes mellitus has been known to man since ancient times and many documents and accounts of experiments are available to explain the origin of this disease. Closer to our times, the classical series of experi-ments in dogs involving pancreatectomies concluded that complete removal of the pancreas caused diabetes (von Mering and Minkowski, 1889). It was not a transient glycosuria but a true, lasting diabetes mellitus which, in every aspect, mimicked the worst type of this disease found in man. It was also concluded that diabetes mellitus was associated with an hitherto unknown function of the pancreas. The possible relationship to the islets of Langerhans did not become evident until several years later. In fact, although pathologists in the early years of this century reported morphologi-cal alterations in the islets of Langerhans of patients with diabetes, it was not until Gepts (1965), by careful morphological investigation, showed that major abnormalities occurred in a number of beta cells and the pres-ence of inflammatory cells in the islets of Langerhans in newly diagnosed Type 1 (insulin-dependent) diabetic patients. Recent studies with morpho-metric techniques (Rahier et al., 1983; Foulis and Stewart, 1984), which allow a quantitative measure of both the total volume or mass of the endo-crine pancreas of individual cell types have shown that Type 1 diabetes in man is associated with a specific loss of the pancreatic beta cells. The disappearance of beta cells is linked to a diminished mass of the endo-crine pancreas, but the total mass of A (glucagon), D (somatostatin) and pancreatic polypeptide cells seemed to remain within the normal range (Rahier et al., 1983). Type 1 diabetes seems, therefore, to represent 2 n a t u r e ' s o w n b e t a - c y t e c t o m y , a p r o c e d u r e that is poss ib le to c a r r y out us ing c h e m i c a l agents l i k e a l l o x a n or s t r e p t o z o t o c i n . T h e p a n c r e a s is an e l o n g a t e d , p ink or y e l l o w i s h - g r a y g l a n d u l a r o r g a n c o m p o s e d of an e x o c r i n e and an e n d o c r i n e p o r t i o n . T h e e x o c r i n e p o r t i o n o f the p a n c r e a s c o m p r i s e s about 98% of the o r g a n . T h e e n d o c r i n e p o r t i o n c o n s i s t s of n u m e r o u s t i n y , h i g h l y v a s c u l a r c l u s t e r s c a l l e d i s l e t s of L a n g e r -hans . T h e i s l e t s (about 1 m i l l i o n ) a re c o m p o s e d of s e v e r a l t y p e s of e p i t h e -l i a l c e l l s i n c l u d i n g A (alpha) c e l l s and B (beta) c e l l s . M o s t o f the p a n c r e a t i c i s l e t c e l l s (60% - 80%) are of the B t y p e and possess m a n y s m a l l g ranu les and s y n t h e s i z e and s e c r e t e i n s u l i n . T h e i n s u l i n m o l e c u l e is c o m p o s e d o f an A c h a i n and a B c h a i n l i n k e d t o g e t h e r by d i s u l f i d e b r i d g e s . B i o l o g i c a c t i v i t y seems to r e s i d e i n the e n t i r e m o l e c u l a r s t r u c t u r e r a t h e r t h a n s p e c i -f i c a m i n o a c i d groupings w i t h i n the m o l e c u l e . It has long been k n o w n that g l u c o s e s t i m u l a t e s the b e t a - c e l l s of the p a n c r e a s to r e l e a s e i n s u l i n w h i c h t h e n ac t s on t a r g e t c e l l s to p r o m o t e g l u c o s e u t i l i z a t i o n and s t o r a g e . S i n c e i n i t i a l l y o n l y g l u c o s e c o u l d be m e a s u r -e d , th i s s y s t e m was g r o s s l y o v e r s i m p l i f i e d , that i s , h y p e r g l y c e m i a was b e l i e v e d to be due to i n s u l i n d e f i c i e n c y and h y p o g l y c e m i a was due to i n s u l i n excess . W i t h the a v a i l a b i l i t y o f i n s u l i n r a d i o i m m u n o a s s a y s , i t b e c a m e c l e a r tha t o n l y a m i n o r i t y of p a t i e n t s c o n f o r m to th is s c h e m e , a m a j o r i t y o f p a t i e n t s w i t h h y p e r g l y c e m i a s are not a b s o l u t e l y i n s u l i n - d e p e n d e n t and have a n o r m a l or s u p e r n o r m a l c o n c e n t r a t i o n o f c i r c u l a t i n g i n s u l i n . D i a b e t e s m e l l i t u s is the n a m e g i v e n to a he terogenous group o f d i s o r d e r s tha t are c h a r a c t e r i z e d by g l u c o s e i n t o l e r a n c e . P r e v i o u s l y d e f i n e d as a c h r o n i c m u l t i -s y s t e m d i s o r d e r c h a r a c t e r i z e d by h y p e r g l y c e m i a c a u s e d by i n s u l i n i n s u f -f i c i e n c y or i n a d e q u a t e i n s u l i n a c t i o n , the new d e f i n i t i o n r e f l e c t s the l a t e s t 3 r e s e a r c h f i n d i n g s i n e p i d e m i o l o g y , g e n e t i c s , v i r o l o g y , i m m u n o l o g y , and b i o -c h e m i s t r y . D i a b e t e s is c h a r a c t e r i z e d by d isorders in the m e t a b o l i s m o f c a r b o h y d r a t e , p r o t e i n , f a t and i n s u l i n , as w e l l as the s t r u c t u r e and f u n c t i o n o f b l o o d vesse ls . These a b n o r m a l i t i e s a c c o u n t f o r b o t h the a c u t e as w e l l as the c h r o n i c c o m p l i c a t i o n s of the disease (see A d d i s o n - W e s l e y , 1983). N e w r e s e a r c h has i n d i c a t e d m o r e t h a n one cause of d i a b e t e s and a grea t dea l of d i v e r s i t y i n d e f i n i t i o n , e x p r e s s i o n and c o u r s e o f the d isease . A new c l a s s i f i c a t i o n s y s t e m was a d o p t e d i n 1979 and T a b l e 1 s u m m a r i z e s the m a j o r c a t e g o r i e s , c u r r e n t t e r m i n o l o g y , o l d labels and m a j o r c l i n i c a l c h a r a c t e r i s t i c s . A s t a b l e 1 i n d i c a t e s , the e t i o l o g y of the disease is not c o m p l e t e l y u n d e r s t o o d and t h e r e are p r o b a b l y m u l t i p l e e t i o l o g i e s w i t h i n e a c h t y p e . In t y p e 1, i t is f e l t t h a t g e n e t i c s , v i r u s e s a n d / o r an a u t o i m m u n e response , a lone or i n c o m b i n a t i o n , are i n v o l v e d . R e c e n t w o r k done w i t h the h u m a n l e u k o c y t e a n t i g e n ( H L A ) s y s t e m t h a t is used in t i ssue t y p i n g has d e m o n s t r a t e d a p o s i t i v e r e l a t i o n s h i p w i t h s o m e H L A ant igens and d i a b e t e s . It has been suggested t h a t the ant igens are d e t e r m i n a n t s of the m o l e c u l a r n a t u r e of c e r t a i n b e t a - c e l l s u r f a c e p r o t e i n s , s o m e o f w h i c h m a y h a v e c e r t a i n unique d e f e c t s tha t r e n d e r the c e l l m o r e s u s c e p t i b l e to c e r t a i n i n s u l t s ( C u d w o r t h and W o o d r o w , 1975; N e r u p , 1978). T h e c h a r a c -t e r i s t i c a l l y abrupt a p p e a r a n c e o f t y p e 1 d i a b e t e s c o u l d a lso be the r e s u l t o f an i n f e c t i o n w i t h a d i a b e t o g e n i c v i r u s . A v i r u s s i m i l a r to , but not i d e n t i -c a l w i t h , the c o x s a c k i e v i r u s has been i d e n t i f i e d ( Y o o n et a l . , 1979); th is v i r u s a t t a c k s and s i g n i f i c a n t l y damages the p a n c r e a t i c b e t a c e l l s . A u t o i m m u n e f a c t o r s m a y also be i m p o r t a n t i n the type 1 f o r m of the d isease . S e r u m f r o m n e w l y d iagnosed t y p e 1 - onset d i a b e t i c s c o n t a i n s an a n t i b o d y c a p a b l e o f c o m p l e x i n g w i t h b e t a c e l l s in the p a n c r e a t i c i s l e t s 4 T A B L E I C L A S S I F I C A T I O N O F D I A B E T E S A N D R E L A T E D G L U C O S E I N T O L E R A N C E S N e w C a t e g o r y T y p e I: I n s u l i n -dependent d i a b e t e s m e l l i t u s ( I D D M ) . 5%-10% of a l l d i a b e t i c s T y p e 2: N o n - i n s u l i n dependent d i a b e t e s ( N I D D M ) . 9 0 % - 9 5 % of o f a l l d i a b e t i c s N o n - o b e s e - 20% of T y p e 2; Obese - 80% of T y p e 2 O l d N a m e J u v e n i l e d i a b e t e s J u v e n i l e - o n s e t d i a b e t e s K e t o s i s - p r o n e d i a b e t e s B r i t t l e d i a b e t e s A d u l t - o n s e t d i a b e t e s M a t u r i t y - o n s e t K e t o s i s - r e s i s t a n t d i a b e t e s S t a b l e d i a b e t e s M a t u r i t y - o n s e t d i a b e t e s o f y o u t h ( M O D Y ) C l i n i c a l C h a r a c t e r i s t i c s - A n y age but u s u a l l y y o u n g M o s t l y t h i n at d iagnos is - C a u s e s m a y be g e n e t i c or v i r a l but p r o b a b l y i n v o l v e a b n o r m a l i m m u n e responses - O f t e n have i s l e t c e l l s a n t i b o d i e s - L i t t l e or no endogenous i n s u l i n - N e e d i n j e c t i o n s to p r e s e r v e l i f e - K e t o s i s prone - A n y age but u s u a l l y o v e r 40, o c c a s i o n a l l y under 21 - C a u s e m a y be g e n e t i c or v i r a l - M o s t l y obese at d iagnos is - N o i s l e t c e l l a n t i b o d i e s - V a r y i n g a m o u n t s of endogenous i n s u l i n , o f t e n h i g h e r t h a n n o r m a l l e v e l s present - M a y need i n s u l i n to a v o i d h y p e r g l y c e m i a ^ R a r e ke tos i s e x c e p t in s t ress or- i n f e c t i o n : I m p a i r e d g luc ose t o l e r a n c e (IGT) A s y m p t o m a t i c d i a b e t e s C h e m i c a l d i a b e t e s S u b c l i n i c a l d i a b e t e s B o r d e r l i n e d i a b e t e s L a t e n t d i a b e t e s - B l o o d g l u c o s e l e v e l s b e t w e e n n o r m a l and that o f d i a b e t i c s - A b o v e n o r m a l s u s c e p t i b i l i t y to a t h e r o s c l e r o t i c d isease - R e n a l and r e t i n a l c o m p l i -c a t i o n s u s u a l l y not s i g n i f i c a n t T A B L E I, c o n t i n u e d N e w C a t e g o r y O l d N a m e s C l i n i c a l C h a r a c t e r i s t i c s G e s t a t i o n a l G e s t a t i o n a l - B e g i n s or is r e c o g n i z e d d i a b e t e s ( G D M ) d i a b e t e s d u r i n g or a f t e r p r e g n a n c y - A b o v e n o r m a l r i s k of p e r i n a t a l c o m p l i c a t i o n s - G l u c o s e i n t o l e r a n c e t r a n s i t o r y but f r e q u e n t l y r e c u r s - 5 0 % go on to d e v e l o p o v e r t d i a b e t e s w i t h i n 15 y e a r s - 8 0 % go on to d e v e l o p o v e r t d i a b e t e s a f t e r 20 y e a r s - A c c o m p a n i e d by c o n d i t i o n s k n o w n or s u s p e c t e d t o cause the d isease - c e r t a i n g e n e t i c s y n d r o m e s D i a b e t e s S e c o n d a r y m e l l i t u s d i a b e t e s a s s o c i a t e d w i t h o t h e r c o n d i t i o n s or s y n d r o m e s F r o m B r u n n e r and S u d d a r t h , 1984 6 o f n o n d i a b e t i c , n o r m a l i n d i v i d u a l s . T h i s a n t i b o d y is f o u n d i n up to 8 5 % of t y p e 1 cases at the t i m e of d iagnos is ( L e r n m a r k et a l . , 1978). O n the o t h e r h a n d , c u r r e n t r e s e a r c h s t r o n g l y suggests tha t the type 2 disease o r i g i -nates as a r e s u l t o f some r e c e p t o r d e f e c t , e i t h e r due to a d e c r e a s e d a f f i n i t y o f the r e c e p t o r s f o r i n s u l i n , a "post r e c e p t o r d e f e c t " (Bar et a l . , 1978) or a " d o w n - r e g u l a t i o n " o f the i n s u l i n r e c e p t o r s in response to p e r m a n e n t l y i n c r e a s e d l e v e l s of i n s u l i n ( K a h n et a l . , 1977). S o m e o t h e r c l a s s i f i c a t i o n s of the d isease are s u m m a r i z e d in T a b l e 1. B . P A T H O P H Y S I O L O G Y O F D I A B E T E S . T h e p a t h o p h y s i o l o g y of d i a b e t e s m e l l i t u s i n c l u d e s i m m e d i a t e e f f e c t s , most o f w h i c h are r e v e r s i b l e w i t h i n s u l i n t h e r a p y , and l o n g - t e r m e f f e c t s , w h i c h g e n e r a l l y appear to t a k e an i n e x o r a b l e c o u r s e . W h a t e v e r the m e c h a n -i s m , i n s u l i n d e p r i v a t i o n of i n s u l i n - d e p e n d e n t c e l l s leads to a m a r k e d d e c r e a s e i n the c e l l u l a r r a t e of g lucose u p t a k e . T h i s r e s u l t s in an i n c r e a s e i n the c o n c e n t r a t i o n o f b l o o d g l u c o s e . T h e h i g h b l o o d g lucose l e v e l s r e s u l t in e l e v a t e d l e v e l s of g l y c o s y l a t e d h e m o g l o b i n s . S i n c e the m o d i f i c a t i o n o f h e m o g l o b i n , w h i c h is due to n o n - e n z y m a t i c g l y c o s y l a t i o n of a m i n o ac ids i n the h e m o g l o b i n m o l e c u l e s , o c c u r s c o n t i n u a l l y d u r i n g the l i f e span of the e r y t h r o c y t e , g l y c o h e m o g l o b i n l e v e l s c a n p r o v i d e a t i m e - a v e r a g e d index o f the degree of h y p e r g l y c e m i a d u r i n g the p r e v i o u s f e w w e e k s d e p e n d i n g on the spec ies in c o n s i d e r a t i o n ( M a c D o n a l d and D a v i s , 1979). A d d i t i o n a l e f f e c t s o f i n s u l i n d e p r i v a t i o n are s u m m a r i z e d in F i g u r e 1. T h e long t e r m p a t h o p h y s i o l o g i c c o m p l i c a t i o n s o f d i a b e t e s m e l l i t u s i n c l u d e r e t i n o p a t h y and c a t a r a c t f o r m a t i o n ( D a v i s , 1974), n e p h r o p a t h y ( B a l o -d i m o s , 1971), n e u r o p a t h y (S ib ley , 1982) and c a r d i o v a s c u l a r diseases w h i c h 7 F I G U R E 1 T h e a c u t e e f f e c t s of i n s u l i n d e p r i v a t i o n . r I Adipose tissue | [ Uver Ketone (brm.uon-«oeu> tceic tod and beta-hyd'O'vbuivnc acid  | Ketones L m blood t-Insultn depnvatxm Depressed tarry acid and triglyceride syntnesis Decreased entry of glucose into instArv dependem cells Increased aver output & glucose j KETOACIDOSIS 6 ram Hyperpnw T ~ ~ r ~ , HYPERGLYCEMIA -•>• ± Caiabolism exceeds enabolism  Excessive release ol tree Utilised by skeletal tarry tods from adipose andcarckacmusde tissue kx energy metabolism Renal threshold for glucose reebsorpuon exceeded * •\ fatty tMiHrauon "j I Amino acids released mio blood T • Hunger | (GLYCOSURIA] =3I Waterless (POLYURIA) Energy metabolism n K>meextrehepauc essue-muscle Salt loss lag, NaCli Lung and Acetone Fruity odor bladder tormetwn to bream | POLYDIPSIA I I L/ve< , T | Daamination | l r = t L G^ coneogenesis ! Ketones Reduced btood volume Decreased oonstncuon of small blood vessels •J, SHOCK | Lethargy L I COMA AND DEATH kvreased non-protein ratrogen •murine Negative nitrogen balance 9 w i l l be d i scussed l a t e r . M o s t o f these e f f e c t s , in b o t h T y p e 1 and T y p e 2 f o r m s of the disease , appear to have as a c o m m o n f e a t u r e the p r o g r e s s i v e t h i c k e n i n g of the b a s e m e n t m e m b r a n e o f c a p i l l a r i e s in the a f f e c t e d r e g i o n s . T h e b a s e m e n t m e m b r a n e s are t h i c k e n e d by a m a t e r i a l tha t appears to be a g l y c o p r o t e i n . T h e f o r m a t i o n of these depos i t s is b e l i e v e d to be the r e s u l t o f g lucose bonding c h e m i c a l l y w i t h a m i n o groups o f body p r o t e i n s , or p o s s i b l y the p r e s e n c e o f i n s u l i n a n t i b o d i e s r e s u l t i n g f r o m the use of beef or p o r c i n e i n s u l i n . R e g a r d l e s s o f the m e c h a n i s m , t h i c k e n i n g o f the c a p i l l a r y w a l l o c c u r s , t i ssue p e r f u s i o n is d i m i n i s h e d and m a n y organs are a f f e c t e d ( for r e v i e w see J a r r e t t , 1984). C . D R U G S A N D C H E M I C A L S T H A T P R O D U C E D I A B E T E S . Insul in s e c r e t i n g c e l l s appear to be m o r e s e n s i t i v e to c h e m i c a l insu l t t h a n o t h e r h o r m o n e - s e c r e t i n g c e l l s of the p a n c r e a s . K n o w l e d g e that c h e m i -c a l s c a n p r o d u c e d i a b e t e s i n l a b o r a t o r y a n i m a l s is l o n g - s t a n d i n g . In the e a r l y 1940s, i t was f o u n d that a l l o x a n , a c y c l i c u r e a a n a l o g , p r o d u c e d a p e r m a n e n t h y p e r g l y c e m i a i n r a b b i t s . T h i s e f f e c t was c a u s e d by the s e l e c t i v e d e s t r u c t i o n o f the i n s u l i n - s e c r e t i n g b e t a - c e l l s o f the e n d o c r i n e p a n c r e a s . T h e m e t h y l n i t r o s u r i a a n a l o g , s t r e p t o z o t o c i n , has h o w e v e r r e p l a c e d a l l o x a n as the p r i m a r y c o m p o u n d used to p r o d u c e e x p e r i m e n t a l , i n s u l i n - d e p e n d e n t d i a b e t e s i n l a b o r a t o r y a n i m a l s . T h i s is due to : 1) the l o w e r m o r t a l i t y r a t e seen in s t r e p t o z o t o c i n - i n d u c e d d i a b e t i c a n i m a l s ( H o f t i e z e r and C a r p e n t e r , 1973), 2) the g r e a t e r s e l e c t i v i t y of s t r e p t o z o t o c i n f o r b e t a c e l l s ( Junoud et a l . , 1969) and 3) the longer h a l f l i f e o f s t r e p t o z o t o c i n i n the body ( A g a r a w a l , 1980). A l t h o u g h these t w o agents share the a b i l i t y to p r o d u c e b e t a - c e l l n e c r o s i s a f t e r s ing le doses in l a b o r a t o r y a n i m a l s , the 10 m e c h a n i s m s i n v o l v e d in th is e f f e c t are d i f f e r e n t . S t r e p t o z o t o c i n c o n t a i n s a N - m e t h y l n i t r o s o u r e a m o i e t y and is an a l k y l a t i n g agent . T h i s p r o p e r t y is c e r t a i n l y c a p a b l e of i n i t i a t i n g i t s c y t o t o x i c a c t i o n . It is r e a s o n a b l e to suggest that the g luc ose m o i e t y present in the m o l e c u l e d i r e c t s the a l k y l a t i n g agent to the p a n c r e a t i c b e t a - c e l l w h i c h m a y have a s p e c i a l a f f i -n i t y f o r g l u c o s e . In s p i t e o f the f a c t tha t a unique r e c e p t o r f o r g lucose i n b e t a c e l l s has not been d e m o n s t r a t e d , i t has been poss ib le to show that s t r e p t o z o t o c i n is c o n c e n t r a t e d in p a n c r e a t i c i s l e t s . T h i s c o n c e n t r a t i n g a c t i o n m a y a c c o u n t for the c e l l - s e l e c t i v e a c t i o n o f the a l k y l a t i n g agent . A l l o x a n is r a p i d l y r e d u c e d i n the body to f o r m d i a l u r i c a c i d , and that p r o d u c t undergoes a u t o - o x i d a t i o n to y i e l d d e t e c t a b l e a m o u n t s o f I ^ C ^ , 2 s u p e r o x i d e a n i o n CO^) and h y d r o x y l f r e e - r a d i c a l s (*OH) . T h e s e r e d u c e d spec ies of o x y g e n , p a r t i c u l a r l y the e x t r e m e l y r e a c t i v e * O H r a d i c a l a re b e l i e v e d to i n i t i a t e the a l l o x a n - b a s e d a t t a c k on b e t a - c e l l s . A l l o x a n and i t s m e t a b o l i t e s have also been shown to c o n c e n t r a t e in p a n c r e a t i c i s l e t t i ssue r e l a t i v e to s o m e o t h e r t i ssues . B e c a u s e a l l o x a n p a r t i c i p a t e s i n the g e n e r a t i o n o f h y d r o g e n p e r o x i d e and the r e a c t i v e f r e e - r a d i c a l s p e c i e s o f o x y g e n , i t is poss ib le tha t the a l l o x a n - s e n s i t i v e b e t a - c e l l c o n t a i n s u n u s u a l l y l o w a m o u n t s of the p r o t e c t i v e subs tances that are present i n a l l c e l l s f o r the purpose o f d e a c t i v a t i n g r e d u c e d f o r m s o f o x y g e n . O f the p r o t e c t i v e subs tances t h a t h a v e been m e a s u r e d , o n l y g l u t a t h i o n e p e r o x i d a s e appears to be low in p a n c r e a t i c i s l e t t i s sue r e l a t i v e to o t h e r t i ssues ( for r e v i e w see F i s c h e r , 1985). D . D I A B E T I C C A R D I O M Y O P A T H Y . D i a b e t e s m e l l i t u s has a m a j o r i m p a c t on c a r d i a c m o r b i d i t y and m o r t a l -11 i t y and c a r d i o v a s c u l a r disease n o w a c c o u n t s f o r a p p r o x i m a t e l y 80% of a l l d i a b e t i c deaths ( K a n n e l , 1978). In the a n i m a l m o d e l s of s e v e r e d i a b e t e s , i s o l a t e d w o r k i n g h e a r t s f r o m c h r o n i c a l l y d i a b e t i c r a t s s h o w e d a d e c r e a s e d a b i l i t y to respond to i n c r e a s e d f i l l i n g pressures and i n c r e a s i n g a f t e r l o a d s ( P e n p a r g k u l et a l . , 1980a; V a d l a m u d i et a l . , 1982). C a r d i a c o u t p u t (at h i g h f i l l i n g pressures) and r e l a x a t i o n r a t e w e r e depressed as w e l l . S i m i l a r l y , i s o l a t e d p a p i l l a r y m u s c l e f r o m d i a b e t i c r a t s had a depressed v e l o c i t y o f s h o r t e n i n g and a d e l a y e d onset o f r e l a x a t i o n ( F e i n et a l . , 1980). F i n a l l y , d e t e r i o r a t i o n o f c a r d i a c f u n c t i o n was seen i n d i a b e t i c r a t s i n the p r e s e n c e o f i n c r e a s i n g a f t e r - l o a d s ( Ingebretsen et a l . , 1980). In e x p e r i m e n t a l s tudies w i t h a n i m a l s o t h e r t h a n r a t s , R e g a n et a l . (1974) us ing 1-year a l l o x a n d i a b e t i c dogs s h o w e d v e n t r i c u l a r s t i f f e n i n g a s s o c i -a t e d w i t h s h o r t e n i n g of l e f t v e n t r i c u l a r e j e c t i o n t i m e . R e g a n ' s group a lso s t u d i e d c h r o n i c a l l y d i a b e t i c R h e s u s m o n k e y ' s and f o u n d s i m i l a r r e s u l t s ; c h r o n i c d i a b e t e s r e s u l t s i n d i m i n i s h e d v e n t r i c u l a r c o m p l i a n c e , l e a d i n g to u n d e r f i l l i n g and this a p p e a r e d to be a r e s u l t o f i n c r e a s e d i n t e r s t i t i a l g l y c o -p r o t e i n and c o l l a g e n ( H a i d e r et a l . , 1981). L e e and D o w n i n g (1979) s t u d i e d the h e m o d y n a m i c s of a c u t e l y d i a b e t i c l ambs and n o t e d no depress ion i n l e f t v e n t r i c u l a r f u n c t i o n . O f i n t e r e s t , h o w e v e r , was the f i n d i n g t h a t c o r o n a r y f l o w was d i m i n i s h e d as a r e s u l t of i n c r e a s e d c o r o n a r y v a s c u l a r r e s i s t a n c e . F i n a l l y , F e i n et a l . (1985b) e x a m i n e d p a p i l l a r y m u s c l e f u n c t i o n i n r a b b i t s m a d e d i a b e t i c w i t h a l l o x a n and f o u n d that the d u r a t i o n of i s o m e t r i c and i s o t o n i c c o n t r a c t i o n and r e l a x a t i o n was m a r k e d l y p r o l o n g e d . In h u m a n s tudies , H a m b y et a l . (1974), i n 16 d i a b e t i c p a t i e n t s , f o u n d l e f t v e n t r i c u l a r d i l a t a t i o n and h y p e r t r o p h y in the absence of s y s t e m i c h y p e r -t e n s i o n or o b s t r u c t i v e l a r g e - c o r o n a r y a r t e r y d isease . R e g a n et a l . (1977) 12 d e s c r i b e d the h e m o d y n a m i c f i n d i n g s in f o u r adul t d i a b e t i c s w i t h h e a r t f a i l u r e and f o u n d b o t h l e f t v e n t r i c u l a r s y s t o l i c and d i a s t o l i c d y s f u n c t i o n w i t h o u t e v i d e n c e of on g o i n g m i c r o v a s c u l a r i s c h e m i a . Independent o f the d u r a t i o n o f d i a b e t e s , d i a b e t i c m e n u n d e r g o i n g supine e x e r c i s e had a l o w e r c a r d i a c o u t p u t d u r i n g e x e r c i s e t h a n d i d c o n t r o l s , a r e s u l t of l o w e r s t r o k e v o l u m e ( K a r l e f o r s , 1966). D i a b e t e s was a lso a s s o c i a t e d w i t h a b n o r m a l i t i e s o f l e f t v e n t r i c u l a r c o m p l i a n c e r e f l e c t e d i n s h o r t e n i n g of the l e f t v e n t r i c u l a r e j e c t i o n t i m e and p r o l o n g a t i o n of the p r e - e j e c t i o n p e r i o d ( A h m e d et a l . , 1975). I n i t i a l l y i t was thought that the c a r d i o v a s c u l a r disease was due to the h i g h i n c i d e n c e o f a t h e r o s c l e r o s i s a f f e c t i n g la rge a r t e r i e s and c a p i l l a r i e s . R e c e n t l y , h o w e v e r , e x p e r i m e n t a l s tudies have sugges ted t h a t a s p e c i f i c c a r d i o m y o p a t h y m a y be a c a u s a l f a c t o r in p r o d u c i n g the i n c r e a s e in m o r t a -l i t y and m o r b i d i t y o f d i a b e t e s ( F e i n et a l . , 1980). T h e pathogenes is o f th i s d i a b e t i c c a r d i o m y o p a t h y is c o m p l e x and m a y i n v o l v e a depressed m y o -c a r d i a l m y o s i n and a c t o m y o s i n C a ^ + - A T P a s e ( M a l h o t r a et a l . , 1981) and s a r c o p l a s m i c r e t i c u l a r C a 2 + - M g 2 + - A T P a s e ( L o p a s c h u k et a l . , 1983), a s h i f t i n g in the m y o s i n i s o e n z y m e d i s t r i b u t i o n so that the i s o e n z y m e a s s o c i a t e d w i t h l o w e r A T P a s e a c t i v i t y p r e d o m i n a t e s ( D i l l m a n , 1980), an i n c r e a s e d m e t a b o l i s m of f a t t y ac ids in the m y o c a r d i a l t i ssue of d i a b e t i c s ( R a n d l e et a l . , 1964) and o t h e r a s s o c i a t e d c a r d i o v a s c u l a r c o n d i t i o n s w h i c h have been r e p o r t e d ( F e i n and S o n n e n b l i c k , 1985a). E . D I A B E T E S A N D A R T E R I A L H Y P E R T E N S I O N . H y p e r t e n s i o n is g e n e r a l l y b e l i e v e d to be m o r e p r e v a l e n t a m o n g d i a b e -t i c s than n o n - d i a b e t i c s ( C h r i s t l i e b , 1973) and is k n o w n to a g g r a v a t e the c a r d i o v a s c u l a r c o m p l i c a t i o n s of d i a b e t e s m e l l i t u s . N o r m o t e n s i v e d i a b e t i c s 13 have less r e t i n a l and r e n a l disease ( K n o w l e r et a l . 1980; P a r v i n g et a l . , 1983) and i n G o l d b l a t t h y p e r t e n s i v e r a t s , c l i p p e d k i d n e y s w e r e p r o t e c t e d f r o m d e v e l o p i n g g l o m e r u l a r les ions w h i l e the u n d i p p e d k i d n e y e x p o s e d to h y p e r t e n s i o n d e v e l o p e d d a m a g e ( M a u e r et a l . , 1978). B l o o d pressure u s u a l l y b e c o m e s e l e v a t e d w h e n n o r m o t e n s i v e r a t s are t r e a t e d w i t h e i t h e r a l l o x a n ( C a v a l i e r e et a l . , 1980) or S T Z (Bunag et a l . , 1982; S a s a k i and B u n a g , 1983; R o d r i g u e s et a l . , 1985). H o w e v e r , the r o l e o f h y p e r t e n s i o n i n the d e v e l o p m e n t of d i a b e t i c c a r d i o m y o p a t h y is not c l e a r . T h e f i r s t s u g g e s t i o n that h y p e r t e n s i o n m a y be c a u s a l l y r e l a t e d to d i a b e t i c c a r d i o m y o -p a t h y c a m e f r o m the e x p e r i m e n t s of F a c t o r et a l . ( F a c t o r et a l . , 1980; F a c t o r et a l . , 1981; F a c t o r et a l . , 1983) on r e n o v a s c u l a r h y p e r t e n s i v e r a t s . T h e y p r o v i d e d e v i d e n c e that the c o m b i n a t i o n of d i a b e t e s m e l l i t u s and h y p e r t e n s i o n p r o d u c e s s i g n i f i c a n t l y g r e a t e r m y o c a r d i a l les ions t h a n w i t h e i t h e r disease a lone , not o n l y at the l i g h t m i c r o s c o p i c l e v e l ( F a c t o r et a l . , 1981) but u l t r a s t r u c t u r a l l y as w e l l ( F a c t o r et a l . , 1983). T h e s e o b s e r -v a t i o n s w e r e s i m i l a r to t h e i r e a r l i e r s tudies on h y p e r t e n s i v e - d i a b e t i c p a t i e n t s ( F a c t o r et a l . , 1980). S u b s e q u e n t l y , t h e y s h o w e d t h a t v e n t r i c u l a r p a p i l l a r y m u s c l e s f r o m h y p e r t e n s i v e - d i a b e t i c r a t s s h o w e d a m a r k e d s l o w i n g o f i s o m e t r i c and i s o t o n i c c o n t r a c t i o n s ( F e i n et a l . , 1984) and that these changes m a y be due to c o r o n a r y m i c r o v a s c u l a r a b n o r m a l i t i e s ( F a c t o r et a l . , 1984). In 1963, O k a m o t o and A o k i i n t r o d u c e d a new m o d e l o f e x p e r i m e n t a l h y p e r t e n s i o n t h a t r e q u i r e d no p h y s i o l o g i c a l , p h a r m a c o l o g i c a l , or s u r g i c a l i n t e r v e n t i o n ( O k a m o t o et a l . , 1963). S i n c e then e x p e r t panels have r e p o r t e d that th i s s p o n t a n e o u s l y h y p e r t e n s i v e r a t is an e x c e l l e n t m o d e l o f e x p e r i -m e n t a l h y p e r t e n s i o n that c o u l d serve as a c o u n t e r p a r t f o r c l i n i c a l e s s e n t i a l 14 h y p e r t e n s i o n ( I L A R N e w s , 1976). M o r p h o l o g i c a l l y s i m i l a r r e s u l t s have been p r e v i o u s l y d e s c r i b e d i n s p o n t a n e o u s l y h y p e r t e n s i v e r a t s i n w h i c h d i a -be tes was p r o d u c e d by a l l o x a n ( H a s h i m o t o , 1969; l a m s and W e x l e r , 1977), as those r e p o r t e d by F a c t o r et a l . i n r e n o v a s c u l a r h y p e r t e n s i v e - d i a b e t i c r a t s . T h e f u n c t i o n a l consequences o f c o m b i n e d h y p e r t e n s i o n and d i a b e t e s i n these a n i m a l s , is h o w e v e r , u n k n o w n . T h e r e f o r e , w e h a v e e x a m i n e d m y o c a r d i a l c o n t r a c t i l e f u n c t i o n in s t r e p t o z o t o c i n d i a b e t i c s p o n t a n e o u s l y h y p e r t e n s i v e a n i m a l s us ing the i s o l a t e d w o r k i n g hear t p r e p a r a t i o n . F . H Y D R A L A Z I N E (an a n t i h y p e r t e n s i v e agent) . H y d r a l a z i n e is a v a s o d i l a t o r used c o m m o n l y for the t r e a t m e n t o f h y p e r t e n s i o n and c o n g e s t i v e hear t f a i l u r e , but the m e c h a n i s m by w h i c h i t e x e r t s i t s v a s o d i l a t i v e a c t i o n has not been c l e a r l y d e f i n e d . H y d r a l a z i n e has been shown to e x e r t a d i r e c t r e l a x a n t e f f e c t on v a s c u l a r s m o o t h m u s c l e (Spokas et a l . , 1983) and this causes a m a r k e d r e d u c t i o n i n s y s t e m i c v a s c u l a r r e s i s t a n c e . A s s y s t e m i c v a s c u l a r r e s i s t a n c e is one of the m a j o r c o m p o n e n t s of a o r t i c i m p e d e n c e , d e c r e a s e d s y s t e m i c v a s c u l a r r e s i s t e n c e is a s s o c i a t e d w i t h an i n c r e a s e i n c a r d i a c o u t p u t and h y d r a l a z i n e has been shown to i m p r o v e c a r d i a c p e r f o r m a n c e ( C o n r a d s o n et a l . , 1984). H o w e v e r , i t has also been sugges ted that h y d r a l a z i n e p r o d u c e s a d i r e c t p o s i t i v e i n o t r o p i c e f f e c t and the i n c r e a s e d c o n t r a c t i l e s t a t e m a y a lso c o n t r i b u t e to i n c r e a s i n g c a r d i a c o u t p u t . Indeed, i n i s o l a t e d c a t p a p i l l a r y m u s c l e p r e p a r a t i o n s (R iggs et a l . , 1978) and r a t i s o l a t e d l e f t a t r i a ( S o n g k i t t i g u n a and R a n d , 1982), a p o s i t i v e i n o t r o p i c e f f e c t o f h y d r a l a z i n e has been o b s e r v e d but o n l y at a v e r y h igh c o n c e n t r a t i o n . A n a s s o c i a t i o n b e t w e e n h y p e r l i p i d e m i a and d i a b e t i c k e t o a c i d o s i s was 15 f i r s t d e m o n s t r a t e d by B l o o r (1916). S i n c e t h e n , b l o o d l i p i d s h a v e been r e p o r t e d to be e l e v a t e d a m o n g a m b u l a n t d i a b e t i c p a t i e n t s . T h e most c o m m o n l y e l e v a t e d f a s t i n g p l a s m a l i p i d l e v e l is t r i g l y c e r i d e ( N i k k i l a , 1984) but the p l a s m a c h o l e s t e r o l c o n c e n t r a t i o n is a lso i n c r e a s e d i n the d i a b e t i c p o p u l a t i o n ( A l b r i n k et a l . 1963) and in S T Z d i a b e t i c r a t s ( R o d r i g u e s e t a l . , 1985). O n e unusual e f f e c t o f h y d r a l a z i n e r e p o r t e d by P e r r y and M i l l s (1962) is i t s a b i l i t y t o l o w e r b l o o d l i p i d s . T h e s e o b s e r v a t i o n s suggest tha t h y d r a l a z i n e m a y h a v e a r o l e in r e g u l a t i n g l i p i d m e t a b o l i s m and c a r d i a c p e r f o r m a n c e i n v i v o . W e t h e r e f o r e i n v e s t i g a t e d the e f f e c t o f h y d r a l a z i n e on e l e v a t e d b l o o d pressure and s e r u m l i p i d s and depressed c a r d i a c c o n t r a c -t i o n i n d i a b e t i c r a t s . G . S E X D I F F E R E N C E S I N T H E S T R E P T O Z O T O C I N M O D E L O F D I A B E T E S . A l t h o u g h the l a b o r a t o r y r a t is a p o p u l a r e x p e r i m e n t a l a n i m a l i n w h i c h to s tudy p h y s i o l o g i c a l and p a t h o l o g i c a l i n t e r v e n t i o n s on c a r d i a c f u n c t i o n , these are s e l d o m s t u d i e d s i m u l t a n e o u s l y i n m a l e and f e m a l e r a t s . In f a c t , sex d i f f e r e n c e s in c a r d i o v a s c u l a r responses to a v a r i e t y o f e x p e r i m e n t a l i n t e r v e n t i o n s h a v e been d o c u m e n t e d (Schaib le et a l . , 1984). T h e m e t h y l n i t r o s o u r e a d e r i v a t i v e o f 2 - d e o x y - g l u c o s e , s t r e p t o z o t o c i n (STZ) , is a v a l u a b l e e x p e r i m e n t a l agent used to p r o d u c e i n s u l i n - d e p e n d e n t d i a b e t e s in a n u m b e r of a n i m a l spec ies , i n c l u d i n g the r a t , w i t h a subsequent d e p r e s s i o n i n c a r d i a c f u n c t i o n ( M c N e i l l , 1985). T h e i n j e c t i o n o f a s ing le dose o f S T Z r e s u l t s i n a dose -dependent r i se in b l o o d g luc o se and a n e a r l y c o m p l e t e d e s t r u c t i o n of the i n s u l i n - s e c r e t i n g b e t a - c e l l s in the Islets o f L a n g e r h a n s . C u r i o u s l y , the h y p e r g l y c e m i c a c t i o n o f S T Z is m a r k e d l y i n f l u e n -c e d by the gender o f the a n i m a l ; m a l e s a r e , in g e n e r a l , s i g n i f i c a n t l y m o r e 16 s u s c e p t i b l e than f e m a l e s ( M c L a r e n et a l . , 1980). A s t r o n g m a l e p r e f e r e n c e has a lso been r e p o r t e d i n the d e v e l o p m e n t of e x p e r i m e n t a l d i a b e t e s i n m i c e a f t e r i n f e c t i o n by a d i a b e t o g e n i c v i r u s ( M o r r o w et a l . , 1980) and i n r a t s t r e a t e d w i t h a l l o x a n ( G o o d m a n and H a z e l w o o d , 1974) or a f t e r n e a r l y t o t a l p a n c r e a t o m y ( R o d r i g u e z , 1965). It s e e m e d poss ib le t h e r e f o r e t h a t the s u s c e p t i b i l i t y to i n s u l i n - d e p e n d e n t d i a b e t e s and d e p r e s s i o n i n c a r d i a c f u n c t i o n in r a t s is gender dependent . In th i s s tudy , w e have assessed l e f t v e n t r i c u l a r f u n c t i o n i n age m a t c h e d m a l e and f e m a l e S T Z d i a b e t i c r a t s us ing the i s o l a t e d w o r k i n g r a t h e a r t p r e p a r a t i o n . T h e m e t h o d a l l o w s q u a n t i f i c a t i o n of m u s c l e f u n c t i o n under w e l l c o n t r o l l e d c o n d i t i o n s in the absence o f s y s t e m i c v a s c u l a r and n e u r o h u m o r a l e f f e c t s 17 S P E C I F I C G O A L S O F T H E P R E S E N T I N V E S T I G A T I O N . 1. T o s t u d y the r o l e o f h y p e r t e n s i o n i n the d e v e l o p m e n t o f d i a b e t i c c a r d i o m y o p a t h y . 2. T o s tudy the f u n c t i o n a l consequences of c o m b i n e d h y p e r t e n s i o n and d i a b e t e s on m y o c a r d i a l c o n t r a c t i l e f u n c t i o n i n S H R d i a b e t i c r a t s . 3. T o l o c a t e a t i m e po in t at w h i c h the m y o c a r d i a l a l t e r a t i o n s in the S H R d i a b e t i c r a t s w e r e g r e a t e r t h a n e i t h e r c o n d i t i o n a l o n e . 4. T o i n v e s t i g a t e the r o l e o f h y d r a l a z i n e i n r e g u l a t i n g l i p i d m e t a b o l i s m , b l o o d pressure and c a r d i a c p e r f o r m a n c e i n d i a b e t i c r a t s . 5. T o e x a m i n e the i n f l u e n c e o f sex d i f f e r e n c e i n c a r d i a c f u n c t i o n i n the S T Z m o d e l of d i a b e t e s . 18 MATERIALS AND METHODS A. MATERIALS 1. CHEMICALS: The following chemicals were purchased from Sigma Chemical Co.: sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium bicarbonate, D-glucose, streptozotocin, 5-hydroxymethylfurfuralde-hyde, hydralazine hydrochloride. Dextrostix were purchased from Ames Co., (Elkhart, Indiana). 2. ASSAY KITS: Glucose assay kits were purchased from Sigma Chemicals. Insulin assay kits were purchased from Amersham. Serum T^ index kits were obtained from Abbot Laboratories. Serum T^ assay kits were obtained from Amersham. Serum phospho-lipids, triglycerides and total cholesterol kits were obtained from Boehrin-ger-Mannheim. B. METHODS 1. INDUCTION OF DIABETES: All animals were transiently anesthetized with ether to allow injec-tion of either streptozotocin (STZ) or its vehicle into the tail vein. Diabetes was induced by a single tail vein injection of streptozotocin (55 mg/kg) dissolved in citrate buffer (pH 4.5). Control rats were injected with citrate buffer alone. All rats injected with STZ survived and were 19 t h e r e a f t e r housed t w o to three per c a g e . F o o d and w a t e r w e r e p r o v i d e d ad l i b i t u m t h r o u g h o u t the s t u d y p e r i o d . S e v e r i t y of the disease was d e t e r -m i n e d by m e a s u r i n g the e x t e n t o f g l y c o s u r i a us ing e n z y m a t i c tes t s t r i p s ( T e s - T a p e , L i l l y ) . T h r e e days a f t e r S T Z i n j e c t i o n , r a t s d i s p l a y i n g g l y c o s u r i a g r e a t e r than 2% w e r e used as the d i a b e t i c g r o u p . 2. T R E A T M E N T P R O T O C O L S a) S I X W E E K S S H R S T U D Y : M a l e ra ts ( n o r m o t e n s i v e W i s t a r 290-300 g; s p o n t a n e o u s l y h y p e r t e n s i v e 225-235 g; C h a r l e s R i v e r C a n a d a , M o n t r e a l , C a n a d a ) w e r e 9 weeks o l d p r i o r to S T Z t r e a t m e n t . F o u r groups o f a n i m a l s w e r e s t u d i e d : W i s t a r c o n t r o l s ( C O N ) ; W i s t a r d i a b e t i c s ( D I A ) ; h y p e r t e n s i v e c o n t r o l s (SHR) and c o m b i n e d h y p e r t e n s i v e and d i a b e t i c s ( S H D ) . T h e e x p e r i m e n t a l p e r i o d was 6 w e e k s , a f t e r w h i c h the a n i m a l s w e r e s a c r i f i c e d . H e a r t s w e r e r e m o v e d and p e r f u s e d as d e s c r i b e d b e l o w . T h e a n i m a l s w e r e k i l l e d 6 w e e k s a f t e r d i a b e t e s i n d u c t i o n s i n c e a l t e r a t i o n s i n c a r d i a c p e r f o r m a n c e o c c u r 6 w e e k s a f t e r the onset of d i a b e t e s ( T a h i l i a n i et a l . , 1983). B l o o d was c o l l e c t e d and a n a l y z e d for g lucose and i n s u l i n . b) T W E L V E W E E K S S H R S T U D Y : M a l e r a t s ( n o r m o t e n s i v e W i s t a r 295-305 g; n o r m o t e n s i v e W i s t a r - K y o t o 235-245 g; s p o n t a n e o u s l y h y p e r t e n s i v e 225-235 g; C h a r l e s R i v e r C a n a d a , M o n t r e a l , C a n a d a ) w e r e 9 weeks o l d p r i o r to S T Z t r e a t m e n t . S i x groups of a n i m a l s w e r e s t u d i e d : W i s t a r c o n t r o l s ( C O N ) ; W i s t a r d i a b e t i c s (DIA) ; 20 W i s t a r - K y o t o c o n t r o l s ( W K C ) ; W i s t a r - K y o t o d i a b e t i c s ( W K D ) ; s p o n t a n e o u s l y h y p e r t e n s i v e c o n t r o l s ( S H C ) ; s p o n t a n e o u s l y h y p e r t e n s i v e d i a b e t i c s ( S H D ) . T h e e x p e r i m e n t a l p e r i o d was 12 w e e k s , a f t e r w h i c h the a n i m a l s w e r e s a c r i -f i c e d . H e a r t s w e r e r e m o v e d and p e r f u s e d as d e s c r i b e d b e l o w . B l o o d was c o l l e c t e d at the t i m e of s a c r i f i c e and a n a l y z e d f o r g l u c o s e , i n s u l i n , T ^ and l i p i d p r o f i l e . A t the end o f e a c h e x p e r i m e n t the r i g h t a t r i a and grea t vessels w e r e d i s s e c t e d f r e e , and the r i g h t v e n t r i c u l a r - f r e e w a l l was r e m o v e d . T h e l e f t v e n t r i c l e ( i n c l u d i n g the septum) was w e i g h e d . It was t h e n d r i e d t o g e t h e r w i t h the r i g h t v e n t r i c l e to a c o n s t a n t w e i g h t i n an o v e n in o r d e r to o b t a i n d r y w e i g h t . c) H Y D R A L A Z I N E S T U D Y : M a l e r a t s ( n o r m o t e n s i v e W i s t a r 200-300 g; C h a r l e s R i v e r C a n a d a , M o n t r e a l , C a n a d a ) w e r e 9 weeks o l d p r i o r to S T Z t r e a t m e n t . T h e r a t s w e r e t h e n r a n d o m l y d i v i d e d i n t o f o u r groups : c o n t r o l s ; h y d r a l a z i n e - t r e a t e d c o n t r o l s ; S T Z - i n j e c t e d (d iabe t i c ) ; and h y d r a l a z i n e - t r e a t e d , S T Z - i n j e c t e d . H y d r a l a z i n e was added to the d r i n k i n g w a t e r of the t r e a t e d a n i m a l s (0.5 to 0.6 m g / m L for the c o n t r o l s , 0.08 to 0.1 m g / m L f o r the d i a b e t i c ) . H y d r a l a z i n e i n t a k e i n m g / k g / d a y was d e t e r m i n e d by the f o l l o w i n g f o r m u l a : 1000 x [average f l u i d i n t a k e per day (mL) x c o n c e n t r a t i o n o f h y d r a l a z i n e ( m g / m L ) ] / [ b o d y w e i g h t (g)] ( Jadhav and J a n d h y a l a , 1983). H e a r t s w e r e r e m o v e d and p e r f u s e d as d e s c r i b e d b e l o w . B l o o d s a m p l e s w e r e c o l l e c t e d f r o m n o n - f a s t i n g a n i m a l s at the t i m e of s a c r i f i c e and assayed f o r g l u c o s e , i n s u l i n , T ^ and T ^ and l i p i d p r o f i l e . A t the end of e a c h e x p e r i m e n t the r i g h t a t r i a and grea t vesse ls w e r e d i s s e c t e d f r e e , and the r i g h t v e n t r i c u l a r f r e e w a l l was r e m o v e d and w e i g h e d . T h e l e f t v e n t r i c l e ( i n c l u d i n g the 21 septum) was also w e i g h e d . d) P R E P A R A T I O N O F I S O L A T E D T I S S U E S : A f t e r s ix w e e k s , an a d d i t i o n a l group o f c o n t r o l and d i a b e t i c r a t s w e r e s t u n n e d by a sharp b l o w to the head and k i l l e d by d e c a p i t a t i o n . T h e hear t s w e r e q u i c k l y r e m o v e d and p l a c e d i n C h e n o w e t h - K o e l l e ( C K ) b u f f e r (pH 7.4), w h i c h was m a i n t a i n e d at 3 7 ° C and c o n s t a n t l y b u b b l e d w i t h c a r b o g e n (95% + 5 % C 0 2 ) . T h e r i g h t and l e f t a t r i a w e r e q u i c k l y d i s s e c t e d out and m o u n t e d in o r g a n baths c o n t a i n i n g C K b u f f e r w h i c h was m a i n t a i n e d under the c o n d i t i o n s m e n t i o n e d a b o v e . R i g h t a t r i a was a l l o w e d to beat s p o n t a n e o u s l y . L e f t a t r i a w e r e s t i m u l a t e d w i t h 5 m s e c square w a v e pulses o f a s u p r a - t h r e s h o l d v o l t a g e at 1 H z us ing a G r a s s s t i m u l a t o r , M o d e l S D - 9 . T h e responses w e r e r e c o r d e d us ing a G r a s s f o r c e d i s p l a c e m e n t t r a n s d u c e r ( M o d e l F T . 0 3 ) w h i c h w a s c o u p l e d to a G r a s s 7 9 D p o l y g r a p h . A r e s t i n g t e n s i o n of 1 g was a p p l i e d to a l l p r e p a r a t i o n s and t h e y w e r e s t a b i l i z e d f o r 60 m i n b e f o r e e x p o s u r e to any d r u g . —6 —3 G r a d e d doses of h y d r a l a z i n e (1x10 to 1x10 M ) w e r e then added i n a c u m u l a t i v e f a s h i o n . T h e c o n t a c t t i m e f o r e a c h dose of h y d r a l a z i n e was 5 m i n . e) M A L E A N D F E M A L E S T U D Y : M a l e and f e m a l e ra ts (male W i s t a r 260-270 g; f e m a l e W i s t a r 170-180 g) w e r e 9 w e e k s o l d p r i o r to S T Z t r e a t m e n t . A t the end of e a c h e x p e r i -m e n t the a t r i a and grea t vesse ls w e r e d i s s e c t e d f r e e , and the r i g h t v e n t r i -22 c u l a r f r e e w a l l was r e m o v e d . T h e l e f t v e n t r i c l e ( i n c l u d i n g the septum) and r i g h t v e n t r i c l e w e r e d r i e d to c o n s t a n t w e i g h t in an o v e n to d e t e r m i n e d r y w e i g h t s . B l o o d c o l l e c t e d was s t o r e d at - 2 0 ° C u n t i l assayed . 3 . W O R K I N G H E A R T P E R F U S I O N . C o n t r o l and d i a b e t i c a n i m a l s w e r e k i l l e d by s t u n n i n g t h e m w i t h a b l o w to the h e a d f o l l o w e d by d e c a p i t a t i o n . H e a r t s w e r e q u i c k l y e x c i s e d f r o m the r a t s and p l a c e d i n w a r m a e r a t e d C h e n o w e t h - K o e l l e s o l u t i o n w h e r e e x t r a n e o u s t i ssue was d i s s e c t e d f r e e . T h e a o r t i c s t u m p was l o c a t e d and t i e d to a 15-gauge s t a i n l e s s s t e e l a o r t i c p e r f u s i o n c a n n u l a . P e r f u s i o n was c a r r i e d out us ing a m o d i f i c a t i o n of the t e c h n i q u e o f N e e l y and R o v e t t o (1975) as d e s c r i b e d by T a h i l i a n i et a l . (1983). P e r f u s i o n w a s i n i t i a t e d i n the r e t r o g r a d e m a n n e r t h r o u g h the a o r t a at 45 c m ^ O (30 m m Hg) a o r t i c f i l l i n g p r e s s u r e . T h e p e r f u s i o n f l u i d used was C h e n o w e t h - k o e l l e b u f f e r m a i n t a i n e d at 3 7 + l ° C and b u b b l e d w i t h c a r b o g e n . T h e m i l l i m o l a r c o n c e n t r a t i o n o f so lu tes i n the b u f f e r w e r e as f o l l o w s : N a C l , 120.0; K C 1 , 5.6; C a C l 2 , 2.18; M g C l 2 , 2 .1 ; N a H C o 3 , 19.2; g l u c o s e , 10. A 16-gauge s t a i n l e s s s t e e l c a n n u l a , c o n n e c t e d to a t r i a l f i l l i n g r e s e r v o i r s , was then i n s e r t e d i n t o and t i e d to the p u l m o n a r y v e i n . L e f t v e n t r i c u l a r d e v e l o p e d pressure was m e a s u r e d by means of a S t a t h a m P 2 3 A A t r a n s d u c e r ( G o u l d S t a t h a m I n s t r u m e n t s Inc.) a t t a c h e d to a 3 c m p i e c e of p o l y e t h y l e n e P E 90 t u b i n g . T h e P E 9 0 tube was a t t a c h e d to a 20-gauge n e e d l e . T h i s was i n s e r t e d t h r o u g h the apex o f the h e a r t i n t o the l e f t v e n t r i c l e . C a r d i a c w o r k was i n i t i a t e d by s w i t c h i n g the p e r f u s i o n s y s t e m f r o m the r e t r o g r a d e m o d e to the w o r k i n g h e a r t m o d e . In the w o r k i n g mode the p e r f u s a t e 23 e n t e r e d the l e f t v e n t r i c l e through the l e f t a t r i u m and was p u m p e d out t h r o u g h the a o r t i c s t u m p . T h e a o r t i c o u t f l o w was s u b j e c t e d to an a f t e r -l o a d o f a 75 c m c o l u m n of 1 ^ 0 , P E 1 6 0 t u b i n g be ing used as the c o l u m n . A n o t h e r S t a t h a m P 2 3 A A t r a n s d u c e r , a t t a c h e d to a s ide a r m of the a o r t i c o u t f l o w s y s t e m , was used to m e a s u r e i n t r a a o r t i c p r e s s u r e . L e f t v e n t r i c u l a r pressure , i n t r a a o r t i c pressure , and the f i r s t d e r i v a t i v e of l e f t v e n t r i c u l a r pressure w e r e r e c o r d e d on a G r a s s m o d e l 79D p o l y g r a p h . E a c h hear t was s t i m u l a t e d by means o f a p l a t i n u m e l e c t r o d e p l a c e d on the l e f t a t r i u m at t w i c e the t h r e s h o l d v o l t a g e w i t h square w a v e pulses o f 6 m s e c d u r a t i o n ( f r o m a G r a s s m o d e l S D 9 D s t i m u l a t o r ) to g i v e a r a t e of 300 b e a t s / m i n . D a t a w e r e c o l l e c t e d and a n a l y z e d w i t h a m i c r o c o m p u t e r ( A p p l e II w i t h a M o u n t a i n h a r d w a r e board) . T h e pressure t r a n s d u c e r s i g n a l f r o m the p o l y g r a p h was s a m p l e d at 667 H z o v e r 1.5 sec at e a c h f u n c t i o n p o i n t . T h i s r e s u l t e d i n d a t a b e i n g c o l l e c t e d f o r s ix c o m p l e t e c a r d i a c pulses . T h r e e o f these w e r e a n a l y z e d us ing c u r v e - f i t t i n g t e c h n i q u e s to d e t e r m i n e pulse h e i g h t , a r e a , s t a r t and f i n i s h . T h e va lues f r o m these t h r e e pulses w e r e a v e r a g e d to p r o d u c e the d a t a v a l u e s at t h a t f u n c t i o n p o i n t T h e hear t s w e r e e q u i l i b r a t e d at 15 c m a t r i a l f i l l i n g pressure for 10 m i n b e f o r e f u n c t i o n c u r v e s w e r e p e r f o r m e d by e s t i m a t i n g the l e f t v e n t r i c u l a r f u n c t i o n against v a r y i n g l e f t a t r i a l f i l l i n g pressures . D i f f e r e n t f i l l i n g pressures w e r e o b t a i n e d by c h a n g i n g the he ight of the l e f t a t r i a l f i l l i n g r e s e r v o i r f r o m 5.0 to 22.5 c m in 2.5 c m s teps . T h e f i l l i n g pressure was f i r s t r e d u c e d s t e p w i s e f r o m 15.0 to 5.0 c m ^ O , a f t e r w h i c h i t w a s i n c r e a s e d s t e p w i s e to 22.5 c m and f i n a l l y d e c r e a s e d s t e p w i s e to 15.0 c m ^ O . A t e a c h p o i n t , pressure d e v e l o p m e n t was a l l o w e d to s t a b i l -i z e b e f o r e i t was r e c o r d e d . In g e n e r a l , s t a b l e pressure d e v e l o p m e n t was 24 a c h i e v e d w i t h i n 2 m i n a f t e r c h a n g i n g the l e f t a t r i a l f i l l i n g pressure . A c o m p l e t e d f u n c t i o n c u r v e was u s u a l l y p e r f o r m e d in about 20-30 m i n . T h e t o t a l t i m e of p e r f u s i o n of e a c h h e a r t was a p p r o x i m a t e l y 45 m i n . 4. M E A S U R E M E N T O F S Y S T O L I C B L O O D P R E S S U R E A N D H E A R T R A T E O F U N A N E S T H E T I Z E D R A T S . F o l l o w i n g p r e t r e a t m e n t w i t h S T Z (or i t s v e h i c l e ) , body w e i g h t , hear t r a t e and s y s t o l i c pressure w e r e m e a s u r e d w e e k l y . T h e t a i l - c u f f m e t h o d was used f o r the d e t e r m i n a t i o n o f s y s t o l i c pressure . T h e m e a s u r e m e n t s w e r e m a d e at the s a m e t i m e o f the day a f t e r w a r m i n g the a n i m a l s at 3 0 ° C f o r 15 m i n p r i o r to p l a c i n g t h e m i n a h e a t e d r a t h o l d e r , a lso m a i n -t a i n e d at 3 0 ° C . S i n c e the a n i m a l s had been p r e c o n d i t i o n e d p r i o r to i n j e c -t i o n o f S T Z they b e c a m e sedate w i t h i n 0.5-2 m i n a f t e r b e i n g p l a c e d i n the r a t h o l d e r . T h e t a i l - c u f f c o n s i s t e d of a m e t a l tube , i n t e r n a l d i a -m e t e r 7/16 i n c h , l i n e d w i t h a t h i n i n f l a t a b l e rubber b l a d d e r 15 m m l o n g . It was p l a c e d at the base of the t a i l and a p n e u m a t i c pulse sensor t a p e d c i r c u m f e r e n t i a l l y to the t a i l d i s t a l to the o c c l u s i o n c u f f . T h e pulse sensor was c o n n e c t e d to a p n e u m a t i c pulse t r a n s d u c e r ( N a r c o B i o S y s t e m s , H o u s t o n , Texas ) . S y s t o l i c pressure was d e t e r m i n e d as the l e v e l at w h i c h p u l s a t i o n s r e a p p e a r e d d u r i n g g r a d u a l d e f l a t i o n o f the c u f f . E a c h m e a s u r e -ment was o b t a i n e d by a v e r a g i n g t h r e e i n d i v i d u a l r e a d i n g s o b t a i n e d i n r a p i d sequence . H e a r t r a t e was c a l c u l a t e d by m u l t i p l y i n g a r t e r i a l p u l s a -t ions r e c o r d e d f o r 5 sees by 12. M a x i m u m c u f f pressure ( m m H g ) , c y c l i n g i n t e r v a l (min) and i n f l a t i o n - d e f l a t i o n r a t e s ( m m H g / s e c ) w e r e k e p t c o n s t a n t us ing a p r o g r a m m e d e l e c t r o s p h y g m o m a n o m e t e r . 25 5. B L O O D A N A L Y S E S : B l o o d c o l l e c t e d was s t o r e d at - 2 0 ° C u n t i l assayed . S e r u m g lucose l e v e l s w e r e d e t e r m i n e d by the g lucose o x i d a s e m e t h o d us ing a S i g m a g l uco se k i t . S e r u m i m m u n o r e a c t i v e i n s u l i n was assayed by the r a d i o -i m m u n o a s s a y m e t h o d us ing the A m e r s h a m i n s u l i n R I A k i t . T h e A m e r l e x T - 4 R I A k i t o f A m e r s h a m was used f o r the r a d i o i m m u n o a s s a y of s e r u m 125 t h y r o x i n e . T h e T ^ B index was d e t e r m i n e d us ing a T r i o b e a d - I u p t a k e k i t o b t a i n e d f r o m A b b o t t l a b o r a t o r i e s . S e r u m was assayed f o r n o n - e s t e r i f i e d f a t t y ac ids by the m e t h o d of K e l l y (1975). S e r u m phospho-l i p i d s , t r i g l y c e r i d e s and t o t a l c h o l e s t e r o l w e r e d e t e r m i n e d us ing B o e h r i n g e r -M a n n h e i m d i a g n o s t i c reagent k i t s . G l y c o s y l a t e d h e m o g l o b i n in b l o o d was assayed by the m e t h o d of S u b r a m a n i a n et a l . (1980). C e l l s o b t a i n e d a f t e r s e p a r a t i o n of p l a s m a w e r e w a s h e d t w i c e w i t h s a l i n e at 4 ° C and w e r e t h e n h e m o l y s e d w i t h d i s t i l l e d water . . T o the r e s u l t a n t s u p e r n a t a n t , a c i d i f i e d a c e t o n e was added to s e p a r a t e out the g l o b i n . T h e g l o b i n p e l l e t o b t a i n e d by c e n t r i f u g i n g the suspension was w a s h e d once w i t h a c i d i f i e d a c e t o n e and then w i t h a c e t o n e to r e m o v e the a c i d . F i n a l l y , the p e l l e t was suspended i n d i e t h y l e t h e r and d r i e d under n i t r o g e n . G l o b i n thus o b t a i n e d was s t o r e d at - 2 0 ° C u n t i l the assay was c a r r i e d out . G l o b i n was assayed by d i g e s t i n g 75 m g i n 4 m L of 10 M a c e t i c a c i d f o r 16 hours at 8 0 ° C . T o a 1 m L a l i q u o t of the digest 1 m L of 2% t r i c h l o r o a c e t i c a c i d was added and the m i x t u r e a l l o w e d to s i t f o r 5 m i n u t e s . T h e suspension was c e n t r i f u g e d and t h i o b a r b i t u r a t e reagent w a s added to the p e l l e t . T h e r e a c t i o n m i x t u r e was i n c u b a t e d f o r 50 m i n u t e s at 4 0 ° C and the absorbance m e a s u r e d at 443 n M . H y d r o x y m e t h y l -26 f u r f u r a l d e h y d e ( H M F ) was used as a s t a n d a r d . T h e degree o f g l y c o s y l a t i o n is e x p r e s s e d as u m o l H M F / g g l o b i n p r o t e i n . 6. S T A T I S T I C A L A N A L Y S E S : A l l r e s u l t s f o r any p a r t i c u l a r group w e r e a v e r a g e d and the s t a n d a r d e r r o r o f the m e a n ( S . E . M . ) d e t e r m i n e d at e a c h e x p e r i m e n t a l p o i n t . O n e w a y ana lys i s o f v a r i a n c e ( A N O V A ) f o l l o w e d by N e w m a n - K e u l ' s tes t was used f o r c o m p a r i n g r e s u l t s f r o m a g i v e n set of groups . A p r o b a b i l i t y o f less than 0.05 (p<0.05) was used as the l e v e l o f s t a t i s t i c a l s i g n i f i c a n c e . 27 R E S U L T S A . S I X W E E K S S H R S T U D Y : I n j e c t i n g a n i m a l s w i t h S T Z r e s u l t e d in a d i a b e t i c s t a t e w i t h an e l e v a t e d u r i n e g luc ose (>2%) throughout the s t u d y p e r i o d . N o d e t e c t a b l e g l u c o s e was present i n the u r i n e of c o n t r o l a n i m a l s . O t h e r s y m p t o m s n o r m a l l y a s s o c i a t e d w i t h the d i a b e t i c s t a t e s u c h as p o l y u r i a , p o l y d i p s i a and p o l y p h a g i a w e r e seen i n the d i a b e t i c r a t s . W e i g h t g a i n d u r i n g the s tudy p e r i o d was s i g n i f i c a n t l y less i n t h e d i a b e t i c t h a n in c o n t r o l a n i m a l s , such t h a t d i a b e t i c r a t s s h o w e d s i g n i f i c a n t l y l o w e r body and h e a r t w e i g h t s a t the t i m e of s a c r i f i c e (Tab le 2). T h e d i f f e r e n c e in t h e w e i g h t of d i a b e t i c ra ts as c o m p a r e d to that o f c o n t r o l r a t s a p p e a r e d t o be due to the loss of fa t depots , e m a c i a t i o n of s k e l e t a l m u s c l e and a s m a l l e r s k e l e t o n . T h e r e was no m o r t a l i t y in any of the d i a b e t i c groups at 6 w e e k s a f t e r t r e a t m e n t . S e r u m g lucose l e v e l s m e a s u r e d at the t i m e o f s a c r i f i c e w e r e m a r k e d l y e l e v a t e d in the S T Z - i n j e c t e d ra ts a c c o m p a n i e d by a d e p r e s s i o n in s e r u m i n s u l i n l e v e l s (Table 2). T a b l e 2 also shows s y s t o l i c b l o o d pressure and hear t r a t e . S y s t o l i c b l o o d pressure i n the D I A r a t p r o g r e s s i v e l y i n c r e a s e d but was s i g n i f i c a n t l y d i f f e r e n t f r o m C O N o n l y on the s i x t h w e e k a f t e r t r e a t m e n t . B y c o n t r a s t S H D r a t s had s y s t o l i c pressures w h i c h w e r e s i m i l i a r to those seen in S H C . B o t h d i a b e t i c groups w e r e o b s e r v e d to show a p r o g r e s s i v e f a l l in hear t r a t e w i t h a v e r a g e s t h r o u g h o u t the s t u d y b e i n g s i g n i f i c a n t l y h igher i n c o n t r o l t h a n in d i a b e t i c r a t s . F i g u r e 2, A - C , i n d i c a t e s the e f f e c t of v a r y i n g f i l l i n g pressures on v a r i o u s p a r a m e t e r s of hear t f u n c t i o n ( le f t v e n t r i c u l a r d e v e l o p e d pressure , +dP/dt , and - d P / d t ) i n d i a b e t i c and c o n t r o l r a t s . S H D ra ts s h o w e d l o w e r T A B L E 2. C E R T A I N F E A T U R E S O F 6 W E E K S C O N T R O L A N D D I A B E T I C R A T S ( S H R study) W i s t a r C o n t r o l n=12 W i s t a r D i a b e t i c n=12 S H R C o n t r o l n=10 S H R D i a b e t i c n=12 B o d y w e i g h t , g 3 4 9 ± 9 H e a r t w t , m g 1 2 7 0 ± 5 0 S e r u m g lucose , 139±9 m g / d L S e r u m i n s u l i n , 39+4 u U / m L S y s t o l i c b lood 138±5 pressure , m m H g H e a r t r a t e , 404± 11 b e a t s / m i n 258+10* 1 0 4 0 ± 3 0 * 4 8 7 ± 1 4 * 1 6 ± 2 * 1 6 1 ± 7 * 330+9* 2 9 8 ± 4 1 3 6 0 ± 2 0 142±8 3 9 ± 2 198+8 3 9 4 ± 2 1 1 9 6 ± 6 * 8 7 0 ± 3 0 * 4 4 9 ± 8 * 1 3 ± 4 * 2 0 9 ± 6 3 3 6 ± 7 * Va lues are mean±SEM; n , number of a n i m a l s . S H R , s p o n t a n e o u s l y h y p e r t e n s i v e . S e r u m glucose and i n s u l i n w e r e d e t e r m i n e d in b l o o d c o l l e c t e d at the t i m e o f d e a t h . B o d y w e i g h t , b lood pressure and hear t r a t e w e r e m e a s u r e d t h r o u g h o u t the 6 w e e k p e r i o d but only the f i n a l va lues t a k e n b e f o r e s a c r i f i c e a re i n d i c a t e d . * S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l of the same s t r a i n at p<0.05. 29 F I G U R E 2 E f f e c t of d i a b e t e s and h y p e r t e n s i o n on (A) l e f t v e n t r i c u l a r d e v e l o p e d pressure , (B) +dP/dt , and (C) - d P / d t of i s o l a t e d p e r f u s e d w o r k i n g h e a r t s at v a r i o u s f i l l i n g pressures . H e a r t s w e r e i s o l a t e d f r o m W i s t a r c o n t r o l ( A - A , n=12) S H R c o n t r o l ( A - A , n=10), W i s t a r d i a b e t i c (•-#>, n=12) and S H R d i a b e t i c ( O - O , n=12) r a t s 42 days a f t e r the i n d u c t i o n of d i a b e t e s w i t h 55 m g / k g i . v . s t r e p t o z o t o c i n . R e s u l t is the m e a n +SE for n u m b e r o f hear ts shown i n parentheses . *p<0.05 vs . c o n t r o l of the s a m e s t r a i n . § p < 0 . 0 5 vs . W i s t a r c o n t r o l . FILLING PRESSURE (cm H2O) 31 l e f t v e n r i c u l a r d e v e l o p e d pressures as c o m p a r e d to t h e i r o w n c o n t r o l s o n l y at the h igher f i l l i n g pressures . In c o n t r a s t , D I A r a t s h a d l e f t v e n t r i c u l a r d e v e l o p e d pressures that w e r e l o w e r than t h e i r o w n c o n t r o l s at a l l f i l l i n g pressures . W h e n S H C was c o m p a r e d to C O N , t h e r e was a l o w e r pressure d e v e l o p e d in S H C at l o w e r f i l l i n g pressures , w h e r e a s at f i l l i n g pressures g r e a t e r t h a n 20 c m H g Q , t h e r e was no d i f f e r e n c e b e t w e e n the t w o groups . S i m i l a r r e s u l t s w e r e also seen for the r a t e of l e f t v e n t r i c u l a r pressure d e v e l o p m e n t (+dP/dt) and the r a t e of pressure d e c l i n e ( - d P / d t ) . T h e r e was no d i f f e r e n c e in c a r d i a c f u n c t i o n b e t w e e n the t w o d i a b e t i c groups . B . T W E L V E W E E K S S H R S T U D Y : V a r i o u s p a r a m e t e r s used to assess the d i a b e t i c s t a t e o f the a n i m a l s w e r e r e c o r d e d and are s u m m a r i z e d i n T a b l e 3. S e r u m g lucose l e v e l s w e r e m a r k e d l y e l e v a t e d i n S T Z - i n j e c t e d r a t s a c c o m p a n i e d by a d e p r e s s i o n i n s e r u m i n s u l i n l e v e l s . M e a s u r e m e n t of n o n - s p e c i f i c g l y c o s y l a t i o n o f h e m o g l o b i n c o n f i r m e d that b l o o d g l u c o s e was e l e v a t e d f o r e x t e n d e d p e r i o d s o f t i m e in the d i a b e t i c r a t s . T h e r e was a h i gh m o r t a l i t y in the S H D a n i m a l s . I n i t i a l l y , w e had 15 a n i m a l s i n th is group, out of w h i c h 7 d i e d (46%) b e f o r e the c o m p l e t i o n of the s t u d y . T h e deaths began a f t e r the s i x t h w e e k and w e r e then e v e n l y d i s t r i b u t e d t h r o u g h o u t the r e m a i n i n g p e r i o d . S e r u m T ^ l e v e l s w e r e s i g n i f i c a n t l y l o w e r in the S H D and D I A r a t s , w h e r e a s the W K D r a t s d i d not show r e d u c t i o n i n T ^ l e v e l s w h e n c o m p a r e d to t h e i r o w n c o n t r o l s . T h e d i a b e t i c r a t s also e x h i b i t e d r e d u c e d body , hear t and l e f t v e n t r i c -u l a r w e i g h t s at 12 w e e k s a f t e r t r e a t m e n t , c o m p a r e d to t h e i r o w n c o n t r o l s T A B L E 3. V A R I O U S P A R A M E T E R S U S E D T O A S S E S S T H E D I A B E T I C S T A T E O F 12 W E E K D I A B E T I C A N I M A L S ( S H R study) C O N n=7 D I A n=8 W K C n=7 W K D n=8 S H C n=7 S H D n=8 S e r u m g lucose , m g / d L S e r u m glucose , i i U / m L G l y c o s y l a t e d h e m o g l o b i n , u m o l H M F / g g l o b i n S e r u m T . P-g/dL M o r t a l i t y 113±4 9 6 ± 1 7 1 .4±0.2 11.69+0.45 4 8 3 ± 2 2 * 2 6 ± 3 * 4 . 9 ± 0 . 4 * 4 . 7 7 ± 0 . 6 1 * 122±6 53+3 1.3±0.2 8 . 5 8 ± 0 . 4 2 4 3 1 ± 1 4 * 2 2 ± 3 * 4 . 5 ± 0 . 4 * 7 . 4 ± 0 . 4 1 112+4 5 7 ± 7 1.6+0.3 1 0 . 4 7 ± 0 . 3 2 4 8 3 ± 1 8 * 28+4* 4 . 6 ± 0 . 4 * 4 . 6 ± 0 . 6 8 * 7 out of 15 46% R e s u l t s are expressed as m e a n ± S E ; n , number of an imals ; C O N , W i s t a r c o n t r o l ; D I A , W i s t a r d i a b e t i c ; W K C , W i s t a r -K y o t o c o n t r o l ; W K D , W i s t a r - K y o t o d i a b e t i c ; S H C , spontaneously h y p e r t e n s i v e c o n t r o l ; S H D , s p o n t a n e o u s l y h y p e r t e n s i v e d i a b e t i c . P a r a m e t e r s w e r e m e a s u r e d in b lood c o l l e c t e d at the t i m e o f d e a t h . D e a t h s in the S H D group began a f t e r the 6 th week and w e r e then e v e n l y d i s t r i b u t e d throughout the r e m a i n i n g p e r i o d . * S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l o f the same s t r a i n at p<0.05. CO to 33 F I G U R E 3 E f f e c t o f d i a b e t e s on body w e i g h t o f S H R (<>-<>> n=8), W i s t a r ( O - O , n=8) and W K Y ( A - A , n=8) d i a b e t i c r a t s a f t e r the i n d u c t i o n of d i a b e t e s w i t h 55 m g / k g i . v . s t r e p t o z o t o c i n . T h e f i g u r e d e m o n -s t r a t e s t h a t , w h e n c o m p a r e d to t h e i r o w n c o n t r o l s , the loss of body w e i g h t in W K Y d i a b e t i c r a t s is not as s e v e r e as the o t h e r t w o d i a b e t i c groups . R e s u l t is the m e a n +SE for n u m b e r of a n i m a l s shown in parentheses . 34 1 M A Q 0 8 Nl 3 S V 3 H 0 3 Q % 35 (Table 4). H o w e v e r , w h e n the W K D r a t s w e r e c o m p a r e d to the o t h e r t w o d i a b e t i c groups , the loss in body w e i g h t was not as p r o f o u n d ( F i g . 3) . D r y h e a r t w e i g h t / b o d y w e i g h t r a t i o s , w h i c h c o r r e c t f o r d i f f e r e n t body w e i g h t s and are thus a m e a s u r e o f r e l a t i v e c a r d i a c h y p e r t r o p h y , w e r e s i g n i f i c a n t l y g r e a t e r i n the D I A and S H D r a t s as c o m p a r e d to t h e i r o w n c o n t r o l s , w h e r e a s th i s r a t i o was s i m i l a r i n the W K C and W K D a n i m a l s , sugges t ing that c a r d i a c h y p e r t r o p h y r e l a t i v e to body w e i g h t had not o c c u r r e d i n the W K D r a t s (Table 4). I n j e c t i n g the a n i m a l s w i t h S T Z r e s u l t e d i n changes in s y s t o l i c a r t e r i a l pressure (SAP) w h i c h are s u m m a r i z e d i n T a b l e 5. S T Z - i n j e c t e d d i a b e t e s had no e f f e c t on S A P of W K D r a t s but i n c r e a s e d the S A P o f D I A r a t s . In the D I A r a t s , S A P was h i g h on the s i x t h w e e k and s t a y e d h i g h t h e r e a f t e r . In c o n t r a s t , S A P of S H D r a t s was r e d u c e d a f t e r 9 w e e k s w h e n c o m p a r e d to t h e i r c o n t r o l s . It a p p e a r e d that the r e d u c t i o n in S A P in these a n i m a l s was due to f a i l u r e i n the p r o g r e s s i v e r i se i n S A P that is seen i n S H C r a t s w i t h t i m e . A t y p i c a l d e c r e a s e i n hear t r a t e d e v e l o p e d in b o t h S H D and D I A r a t s and these w e r e s i g n i f i c a n t l y less t h a n a g e - m a t c h e d c o n t r o l s t h r o u g h o u t the 12-week p e r i o d of s t u d y . In c o n t r a s t , W K D r a t s e x h i b i t e d no s i g n i f -i c a n t d i f f e r e n c e s i n hear t r a t e f r o m t h e i r c o r r e s p o n d i n g c o n t r o l s ( F i g 4) . F i g u r e 5, A - D , shows the s e r u m l i p i d p r o f i l e in c o n t r o l and d i a b e t i c r a t s . S e r u m f r e e f a t t y a c i d s (5A) , t r i g l y c e r i d e s (5B) and t o t a l c h o l e s t e r o l (5C) w e r e s i g n i f i c a n t l y h i g h e r i n S H D and D I A r a t s but t h e r e was no d i f f e r e n c e i n W K D r a t s w h e n c o m p a r e d to t h e i r o w n c o n t r o l s a f t e r 12 w e e k s . S e r u m phosphol ip ids w e r e e l e v a t e d o n l y in the D I A r a t s (5D). T A B L E 4. G E N E R A L C H A R A C T E R I S T I C S O F 12 W E E K C O N T R O L A N D D I A B E T I C R A T S ( S H R study) C O N n=7 D I A n=8 W K C n=7 W K D n=8 S H C n=7 S H D n=8 B o d y w t , 9 5 2 1 ± 1 4 3 2 1 ± 1 1 * 351+6 2 7 6 ± 1 0 * 3 2 2 ± 6 173+9* H e a r t w t , g 1 .33±0 .04 1 . 0 7 ± 0 . 0 2 * 1 .12±0 .02 1 . 0 3 ± 0 . 0 5 * 1.14+0.02 0 . 6 6 ± 0 . 0 2 * L e f t v e n t r i c u l a r w t , g 1 .02±0 .03 0 . 7 9 ± 0 . 0 3 * 0 . 8 9 ± 0 . 0 1 0 . 7 7 ± 0 . 0 3 * 0.91+0.03 0 . 5 3 ± 0 . 0 2 * D r y heart w t , mg 2 1 6 ± 1 6 1 8 1 ± 7 * 209+7 165+11* 189±9 1 1 6 ± 5 * D r y heart w t / B o d y w t , m g / g m 0 . 4 2 ± 0 . 0 2 0.60+0.01* 0 . 6 1 ± 0 . 0 2 0 . 5 6 ± 0 . 0 4 0 . 6 1 ± 0 . 0 3 0 . 7 3 ± 0 . 0 2 * R e s u l t s are expressed as m e a n ± S E ; n , number of a n i m a l s . C O N , W i s t a r c o n t r o l ; D I A , W i s t a r d i a b e t i c ; W K C , W i s t a r -K y o t o c o n t r o l ; W K D , W i s t a r - K y o t o d i a b e t i c ; S H C , spontaneously h y p e r t e n s i v e c o n t r o l ; S H D , s p o n t a n e o u s l y h y p e r t e n s i v e d i a b e t i c . * S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l of the same s t r a i n at p<0.05. cn 37 T A B L E 5. E F F E C T S O F S T R E P T O Z O T O C I N (STZ) P R E T R E A T M E N T O N S Y S T O L I C B L O O D P R E S S U R E ( m m H g ) I N 12 W E E K S C O N T R O L A N D D I A B E T I C R A T S ( S H R study) W e e k s a f t e r S T Z i n j e c t i o n R A T G R O U P -1 +3 +6 +9 + 12 C O N (n=7) 1 3 2 ± 3 136±2 139±5 142±7 150±4 D I A (n=8) 128+4 1 3 4 ± 6 164+5* 1 6 5 ± 7 * 171+7* W K C (n=7) 1 3 4 ± 6 144+3 154+6 157+4 157±4 W K D (n=8) 136±6 139+4 160+7 163+4 165±6 S H C (n=7) 185±3 199+2 203+8 2 3 9 ± 7 2 5 9 ± 7 S H D (n=8) 190±5 195±8 2 2 0 ± 6 1 9 6 ± 9 * 1 9 6 ± 9 * R e s u l t s are e x p r e s s e d as m e a n ± S E ; n , n u m b e r of a n i m a l s . C O N , W i s t a r c o n t r o l ; D I A , W i s t a r d i a b e t i c ; W K C , W i s t a r - K y o t o c o n t r o l ; W K D , W i s t a r - K y o t o d i a b e t i c ; S H C , s p o n t a n e o u s l y h y p e r t e n s i v e c o n t r o l ; S H D , s p o n t a n e o u s l y h y p e r t e n s i v e d i a b e t i c . T a i l - c u f f was used to m e a s u r e s y s t o l i c p r e s s u r e . B y 3 w e e k s a l l our r a t s had g r o w n a c c u s t o m e d to r e p e a t e d t a i l - c u f f m e a s u r e m e n t s so that any stresses i n d u c e d w o u l d have been m i n i m a l . * S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l of the s a m e s t r a i n at p<0.05. 38 F I G U R E 4 E f f e c t o f d i a b e t e s on hear t r a t e o f S H R , W K Y and W i s t a r r a t s . R e s u l t is the m e a n ± S E for n u m b e r of a n i m a l s shown in p a r e n -theses . A s t e r i s k s i n d i c a t e a s i g n i f i c a n t d i f f e r e n c e f r o m c o n t r o l of the s a m e s t r a i n , p<0.05. 40 F I G U R E 5 A S e r u m f r e e f a t t y a c i d s i n S H R , W K Y and W i s t a r r a t s 12 w e e k s a f t e r s t r e p t o z o t o c i n i n j e c t i o n . R e s u l t is the m e a n ± S E f o r c o n t r o l (n=7) and d i a b e t i c a n i m a l s (n=8). A s t e r i s k s i n d i c a t e s i g n i f i -c a n t d i f f e r e n c e f r o m c o n t r o l of the s a m e s t r a i n , p<0.05. WISTAR WISTAR-KYOTO SHR 42 F I G U R E 5B S e r u m t r i g l y c e r i d e s in S H R , W K Y and W i s t a r r a t s 12 w e e k s a f t e r s t r e p t o z o t o c i n i n j e c t i o n . R e s u l t is the m e a n +SE for c o n t r o l (n=7) and d i a b e t i c a n i m a l s (n=8). A s t e r i s k s i n d i c a t e s i g n i f i c a n t d i f f e r e n c e f r o m c o n t r o l of the s a m e s t r a i n , p<0.05. 1000 r • CONTROL (n=7) S3 DIABETIC (n=8) 800 h 600 400 200 * i 1 WISTAR WISTAR-KYOTO SHR 44 F I G U R E 5 C S e r u m t o t a l c h o l e s t e r o l i n S H R , W K Y and W i s t a r r a t s 12 w e e k s a f t e r s t r e p t o z o t o c i n i n j e c t i o n . R e s u l t is the m e a n +SE f o r c o n t r o l (n=7) and d i a b e t i c a n i m a l s (n=8). A s t e r i s k s i n d i c a t e s i g n i f i c a n t d i f f e r -ence f r o m c o n t r o l of the s a m e s t r a i n , p<0.05. WISTAR WISTAR-KYOTO SHR On. 46 F I G U R E 5 D S e r u m p h o s p h o l i p i d s in S H R , W K Y and W i s t a r r a t s 12 w e e k s a f t e r s t r e p t o z o t o c i n i n j e c t i o n . R e s u l t is the m e a n ± S E f o r c o n t r o l (n=7) and d i a b e t i c a n i m a l s (n=8). A s t e r i s k s i n d i c a t e s i g n i f i c a n t d i f f e r -ence f r o m c o n t r o l of the s a m e s t r a i n , p<0.05. fM C0NTR0L(n=7) DIABETIC (n=8) W I S T A R - K Y O T O SHR 48 T h e i n c r e a s e n o t e d in the S H D r a t s was not s i g n i f i c a n t f r o m i t s c o n t r o l . T h e r e w a s no i n c r e a s e i n phosphol ip ids i n W K D r a t s . F i g u r e 6, A - C , i n d i c a t e s the e f f e c t of v a r y i n g f i l l i n g pressures on v a r i o u s p a r a m e t e r s of hear t f u n c t i o n ( le f t v e n t r i c u l a r d e v e l o p e d pressure , +dP/dt and - d P / d t ) i n d i a b e t i c and c o n t r o l r a t s . T h e l e f t v e n t r i c u l a r d e v e l o p e d p r e s s u r e ( L V D P ) was depressed in hear t s f r o m S H D and D I A r a t s , at l e f t a t r i a l f i l l i n g pressures g r e a t e r t h a n 12.5 c m of i n the S H D r a t s and at a l l l e f t a t r i a l f i l l i n g pressures in the D I A r a t s . L V D P i n h e a r t s f r o m W K D r a t s d i d not d i f f e r f r o m t h e i r o w n c o n t r o l s . S i m i l a r -l y , the r a t e o f l e f t v e n t r i c u l a r pressure d e v e l o p m e n t (+dP/dt) and the r a t e of pressure d e c l i n e ( -dP/dt ) w e r e s i g n i f i c a n t l y l o w e r i n S H D and D I A r a t s c o m p a r e d w i t h t h e i r o w n c o n t r o l s , w h e r e a s d iabe tes d i d not a l t e r these p a r a m e t e r s in W K D r a t s . F i g u r e 7 s u m m a r i z e s the c a r d i a c f u n c t i o n in a l l s ix groups of r a t s . T h e L V D P of S H C r a t hear t s was s i g n i f i c a n t l y l o w e r then C O N r a t s at f i l l i n g pressures b e l o w 10 c m ^ O , w h e r e a s no change was o b s e r v e d b e t w e e n the S H C and W K C r a t s . T h e L V D P o f S H D hear t s was the lowes t o f the s ix groups and was s i g n i f i c a n t -l y d i f f e r e n t f r o m the o t h e r t w o d i a b e t i c groups at f i l l i n g pressures g r e a t e r t h a n 12.5 c m ^ O . S i m i l a r l y , the r a t e of l e f t v e n t r i c u l a r pressure d e v e l -o p m e n t (+dP/dt) and the r a t e of pressure d e c l i n e ( -dP/dt ) w e r e s i g n i f i c a n t l y l o w e r i n S H D r a t s c o m p a r e d w i t h a l l o t h e r groups . C . H Y D R A L A Z I N E S T U D Y : 1. G E N E R A L F E A T U R E S O F E X P E R I M E N T A L R A T S . I n j e c t i n g a n i m a l s w i t h S T Z r e s u l t e d i n a d i a b e t i c s t a t e w i t h an e l e v a t e d u r i n e g lucose (>2%) throughout the s t u d y p e r i o d . N o d e t e c t a b l e 49 F I G U R E 6 A E f f e c t o f d i a b e t e s and h y p e r t e n s i o n on l e f t v e n t r i c u l a r d e v e l o p e d pressure o f i s o l a t e d p e r f u s e d w o r k i n g hear t s at v a r i o u s f i l l i n g pressures . H e a r t s w e r e i s o l a t e d f r o m the 6 e x p e r i m e n t a l groups 84 days a f t e r the i n d u c t i o n o f d i a b e t e s w i t h 55 m g / k g i . v . s t r e p -t o z o t o c i n . E a c h po in t r e p r e s e n t s m e a n ± S E f o r n u m b e r of h e a r t s s h o w n i n parentheses . A s t e r i s k s i n d i c a t e s i g n i f i c a n t d i f f e r e n c e f r o m c o n t r o l o f the s a m e s t r a i n , p<0.05. 5 0 « . — - . 1 r-5 10 15 2 0 6 10 16 2 0 2 6 6 10 15 2 0 2 5 FILLING PRESSURE ( O H ^ D ) o 51 F I G U R E 6B E f f e c t of d i a b e t e s and h y p e r t e n s i o n on +dP/dt of i s o l a t e d p e r f u s e d w o r k i n g h e a r t s at v a r i o u s f i l l i n g pressures . H e a r t s w e r e i s o l a t e d f r o m the 6 e x p e r i m e n t a l groups 84 days a f t e r the i n d u c t i o n of d i a b e t e s w i t h 55 m g / k g i . v . s t r e p t o z o t o c i n . E a c h po in t r e p r e s e n t s m e a n ± S E f o r n u m b e r o f h e a r t s shown in parentheses . A s t e r i s k s i n d i c a t e s i g n i f i c a n t d i f f e r e n c e f r o m c o n t r o l of the s a m e s t r a i n , p<0.05. FILLING PRESSURE (Cm H 20) 53 F I G U R E 6 C E f f e c t o f d i a b e t e s and h y p e r t e n s i o n on - d P / d t of i s o l a t e d p e r -fused w o r k i n g h e a r t s at v a r i o u s f i l l i n g pressures . H e a r t s w e r e i s o l a -t e d f r o m the 6 e x p e r i m e n t a l groups 84 days a f t e r the i n d u c t i o n of d i a b e t e s w i t h 55 m g / k g i . v . s t r e p t o z o t o c i n . E a c h po in t r e p r e s e n t s m e a n ± S E f o r n u m b e r of h e a r t s shown i n parentheses . A s t e r i s k s i n d i c a t e s i g n i f i c a n t d i f e r e n c e f r o m c o n t r o l o f the s a m e s t r a i n , p<0.05. 55 F I G U R E 7 E f f e c t s of S T Z - i n d u c e d d i a b e t e s (12 weeks) on m y o c a r d i a l f u n c t i o n o f p e r f u s e d h e a r t s i s o l a t e d f r o m W i s t a r c o n t r o l ( O - O , n=7), S H R c o n t r o l ( • - • , n=7), W K Y c o n t r o l ( A - A , n=7), W K Y d i a b e t i c ( v - y , n=8), W i s t a r d i a b e t i c ( O - O , n=8) and S H R d i a b e t i c ( • - • , n=8) r a t s . L e f t v e n t r i c u l a r d e v e l o p e d pressure ( L V D P ) is p l o t t e d against l e f t a t r i a l f i l l i n g pressures . T h e f i g u r e d e m o n s t r a t e s the s e v e r e d e p r e s s i o n o f L V D P in h y p e r t e n s i v e - d i a b e t i c r a t s . R e s u l t is the m e a n +SE f o r n u m b e r of hear t s shown in parentheses . *p<0.05 vs . c o n t r o l o f the s a m e s t r a i n . p<0.05 vs . a l l o t h e r groups . 56 57 g l u c o s e was present i n the u r i n e o f c o n t r o l a n i m a l s . O t h e r s y m p t o m s n o r m a l l y a s s o c i a t e d w i t h the d i a b e t i c s t a t e such as p o l y u r i a and p o l y p h a g i a w e r e seen i n the d i a b e t i c r a t s . T h e a n i m a l s used i n th i s s t u d y are d e s c r i b e d in T a b l e 6. W e i g h t g a i n d u r i n g the s t u d y p e r i o d was s i g n i f i c a n t l y less i n b o t h d i a b e t i c groups than i n c o n t r o l a n i m a l s so that d i a b e t i c r a t s had s i g n i f i c a n t l y l o w e r body w e i g h t s at the t i m e o f s a c r i f i c e . T h e d i f f e r -ence i n the w e i g h t of d i a b e t i c r a t s as c o m p a r e d to that o f c o n t r o l r a t s a p p e a r e d to be due to the loss of f a t depots , e m a c i a t i o n of s k e l e t a l m u s c l e and a s m a l l e r s k e l e t o n . T h e r e was also a s i g n i f i c a n t d i f f e r e n c e b e t w e e n the w e i g h t s of u n t r e a t e d c o n t r o l and h y d r a l a z i n e - t r e a t e d c o n t r o l a n i m a l s w h i c h g a i n e d w e i g h t m o r e s l o w l y than d i d the c o n t r o l s . D i a b e t i c r a t s e x h i b i t e d r e d u c e d hear t and l e f t v e n t r i c u l a r w e i g h t s w h i c h w e r e r e v e r s e d by h y d r a l a z i n e t r e a t m e n t . L e f t v e n t r i c l e / r i g h t v e n t r i c l e r a t i o s w h i c h are a m e a s u r e of r e l a t i v e l e f t v e n t r i c u l a r h y p e r t r o p h y w e r e s i g n i f i -c a n t l y g r e a t e r o n l y i n the u n t r e a t e d d i a b e t i c group s u g g e s t i n g that c a r d i a c h y p e r t r o p h y had o c c u r r e d i n these r a t s . H e a r t r a t e d e c r e a s e d i n u n t r e a t e d d i a b e t i c r a t s and r e m a i n e d s i g n i f i -c a n t l y less t h a n that o f a g e - m a t c h e d c o n t r o l s throughout the 6 -week p e r i o d of s t u d y . In c o n t r a s t , h y d r a l a z i n e - t r e a t e d d i a b e t i c r a t s e x h i b i t e d no s i g n i f i c a n t d i f f e r e n c e s i n hear t r a t e w h e n c o m p a r e d to e i t h e r c o n t r o l ( F i g . 8). 2. P A R A M E T E R S U S E D T O A S S E S S D I A B E T I C S T A T E O F E X P E R I M E N T A L R A T S : V a r i o u s p a r a m e t e r s used to assess the d i a b e t i c s t a t e of the a n i m a l s w e r e r e c o r d e d and are s u m m a r i z e d i n T a b l e 7. S e r u m g l u c o s e l e v e l s T A B L E 6. G E N E R A L F E A T U R E S O F T H E E X P E R I M E N T A L R A T S ( h y d r a l a z i n e s tudy) C O N T R O L S T R E P T O Z O T O C I N - I N J E C T E D U n t r e a t e d n=8 H y d r a l a z i n e -t r e a t e d n=6 U n t r e a t e d n=8 H y d r a l a z i n e -t r e a t e d n=8 B o d y w e i g h t , gm W e t hear t w e i g h t , gm L e f t v e n t r i c u l a r w e i g h t , gm L e f t v e n t r i c l e / r i g h t v e n t r i c l e (gm/gm) F l u i d i n t a k e , m L / d a y H y d r a l a z i n e i n t a k e , m g / k g / d a y 3 8 4 ± 4 0.997+0.024 0 . 7 8 4 ± 0 . 0 2 4 4 . 2 4 ± 0 . 2 7 30±2 339+4* 0 . 9 9 8 ± 0 . 0 2 5 0.787+0.022 4.35+0.23 35±1 51+3 2 8 5 ± 9 * t 0 . 8 6 3 ± 0 . 0 1 6 t r 0.676+0.011 -ff 5.01+0.18 i t 1 4 5 ± 6 * t 0 2 9 2 ± 1 0 * t 0 . 9 8 6 ± 0 . 0 1 4 0 . 7 6 9 ± 0 . 0 1 7 4 . 4 4 ± 0 . 1 3 157+12* t 5 3 ± 5 B o d y w e i g h t was m e a s u r e d throughout the 6 week p e r i o d but o n l y the f i n a l v a l u e s t a k e n b e f o r e s a c r i -f i c e are i n d i c a t e d . S i n c e d i a b e t i c ra ts drank m o r e w a t e r t h a n c o n t r o l s , the c o n c e n t r a t i o n of h y d r a l a z i n e had to be adjusted w e e k l y to get a f i n a l dose of b e t w e e n 50 t o 55 m g / k g / d a y i n b o t h c o n t r o l and d i a b e t i c t r e a t e d groups . R e s u l t s are expressed as m e a n ± S E ; n , numbers of a n i m a l s ; * s i g n i f i c a n t l y d i f f e r e n t f r o m u n t r e a t e d c o n t r o l s (p<0.05); t s i g n i f i c a n t l y d i f f e r e n t f r o m h y d r a l a z i n e - t r e a t e d c o n t r o l s (p<0.05); t t s i g n i f i -c a n t l y d i f f e r e n t f r o m the o ther three groups (p<0.05). 59 F I G U R E 8 E f f e c t of d i a b e t e s and h y d r a l a z i n e t r e a t m e n t on h e a r t r a t e of e x p e r i m e n t a l r a t s . R e s u l t is the m e a n ± S E f o r the n u m b e r o f a n i m a l s s h o w n in parentheses . A s t e r i s k s i n d i c a t e s i g n i f i c a n t d i f f e r -ences b e t w e e n c o n t r o l vs . d i a b e t i c r a t s , p<0.05. 500 TIME (wk a f te r S T Z i n jec t i on) 61 m e a s u r e d at the t i m e o f s a c r i f i c e w e r e m a r k e d l y e l e v a t e d in the S T Z - i n j e c t e d r a t s a c c o m p a n i e d by a d e p r e s s i o n i n s e r u m i n s u l i n l e v e l s . S e r u m l e v e l s w e r e s i g n i f i c a n t l y l o w e r i n the u n t r e a t e d d i a b e t i c r a t s , w h e r e a s the h y d r a l a z i n e - t r e a t e d d i a b e t i c r a t s d i d not show r e d u c t i o n in l e v e l s w h e n c o m p a r e d to e i t h e r c o n t r o l s . S i m i l a r l y , the T ^ B index was r e d u c e d in the u n t r e a t e d d i a b e t i c a n i m a l s w h i l e the h y d r a l a z i n e - t r e a t e d d i a b e t i c s d i d not e x h i b i t any such d e p r e s s i o n . Thus i t was e v i d e n t tha t S T Z - t r e a t m e n t r e s u l t e d i n a d i a b e t i c s t a t e and that the h y d r a l a z i n e t r e a t -m e n t used a p p e a r e d to be an e f f e c t i v e m e t h o d of p r e v e n t i n g the depressed t h y r o i d s ta tus i n d i a b e t i c r a t s h a v i n g no e f f e c t on the e l e v a t e d g lucose or depressed i n s u l i n l e v e l s . I n j e c t i n g the a n i m a l s w i t h S T Z r e s u l t e d i n changes i n s y s t o l i c a r t e r i a l pressure ( S A P ) w h i c h are s u m m a r i z e d i n T a b l e 8. H y d r a l a z i n e - t r e a t e d c o n t r o l ra ts had l o w e r S A P w h i c h c o n s i s t e n t l y a v e r a g e d about 120 m m H g a f t e r the f o u r t h w e e k of t r e a t m e n t . S A P i n the u n t r e a t e d d i a b e t i c r a t p r o g r e s s i v e l y i n c r e a s e d and was s i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l s by the f i f t h w e e k a f t e r t r e a t m e n t . B y c o n t r a s t , h y d r a l a z i n e - t r e a t e d d i a b e t i c r a t s d i d not show such e l e v a t e d S A P . 3. E F F E C T O F H Y D R A L A Z I N E O N C O N T R O L A N D D I A B E T I C R A T H E A R T S : F i g u r e 9, A - C , i n d i c a t e s the e f f e c t o f v a r y i n g f i l l i n g pressures on v a r i o u s p a r a m e t e r s o f hear t f u n c t i o n ( le f t v e n t r i c u l a r d e v e l o p e d pressure , +dP/dt and - d P / d t ) . H e a r t s f r o m u n t r e a t e d d i a b e t i c ra ts s h o w e d l o w e r l e f t v e n t r i c u l a r d e v e l o p e d pressures , +dP/dt and - d P / d t c o m p a r e d to u n -t r e a t e d c o n t r o l s at a l l f i l l i n g pressures . H y d r a l a z i n e t r e a t m e n t o f the d i a b e t i c r a t s r e s t o r e d the d e v e l o p e d pressure to n o r m a l but o n l y at f i l l i n g T A B L E 7. V A R I O U S P A R A M E T E R S U S E D T O A S S E S S T H E D I A B E T I C S T A T E O F T H E E X P E R I M E N T A L R A T S ( h y d r a l a z i n e study) C O N T R O L S T R E P T O Z O T O C I N - I N J E C T E D U n t r e a t e d n=8 H y d r a l a z i n e -t r e a t e d n=6 U n t r e a t e d n=8 H y d r a l a z i n e -t r e a t e d n=8 S e r u m glucose , m g % 9 9 ± 3 102+6 526+44* t 4 7 8 ± 4 9 * t S e r u m i n s u l i n , y U / m L 48+6 45+5 1 2 ± 2 * t 1 1 ± 1 * t S e r u m T . , Ug/dL 4 . 9 ± 0 . 3 4 . 9 ± 0 . 3 5 2.8+0.3 t t 4.1 ± 0 . 4 4 T „ B index , J % 5 7 . 9 ± 1 . 3 5 7 . 1 ± 1 . 0 47.6+1.9 t t 5 3 . 6 ± 0 . 7 5 P a r a m e t e r s w e r e m e a s u r e d i n b lood c o l l e c t e d at the t i m e o f d e a t h . F o r the i n s u l i n assay, h u m a n i n s u l i n s tandards w e r e used s ince the e r r o r made in the e s t i m a t i o n of r a t i n s u l i n was m i n i -m a l o w i n g to the v e r y h igh cross r e a c t i v i t y w i t h ra t i n s u l i n (about 90%). T ^ B i n d e x shows the e x t e n t of excess b i n d i n g of t h y r o x i n e b i n d i n g g l o b u l i n . R e s u l t s a re e x p r e s s e d as mean+SE; n , n u m b e r of an imals ; * s i g n i f i c a n t l y d i f f e r e n t f r o m u n t r e a t e d c o n t r o l s (p<0.05); t s i g n i f i c a n t l y d i f f e r e n t f r o m h y d r a l a z i n e - t r e a t e d c o n t r o l s (p<0.05); t t s i g n i f i c a n t l y d i f f e r e n t f r o m the o t h e r t h r e e groups (p<0.05). 63 T A B L E 8. E F F E C T S O F S T R E P T O Z O T O C I N (STZ) P R E T R E A T M E N T O N S Y S T O L I C B L O O D P R E S S U R E ( m m H g ) O F C O N T R O L A N D D I A B E T I C R A T S ( h y d r a l a z i n e study) W E E K S A F T E R S T Z I N J E C T I O N -1 +2 +4 +5 +6 U n t r e a t e d c o n t r o l (n=8) 130+2 1 4 2 ± 4 1 4 2 ± 3 134±4 1 3 8 ± 5 H y d r a l a z i n e - t r e a t e d c o n t r o l (n=6) 127+2 1 2 8 ± 4 128±6 1 1 9 ± 3 * 1 1 9 ± 4 * U n t r e a t e d d i a b e t i c (n=8) 138+3 131±4 1 4 8 ± 1 6 1 5 3 ± 5 t 161±7 t H y d r a l a z i n e - t r e a t e d d i a b e t i c (n=8) 1 3 3 ± 4 1 2 5 ± 2 137±6 127±5 123+3 T a i l - c u f f m e t h o d was used t o m e a s u r e s y s t o l i c p r e s s u r e . B y 3 w e e k s a l l our r a t s had g r o w n a c c u s t o m e d to r e p e a t e d t a i l - c u f f m e a s u r e m e n t s so that any s t resses i n d u c e d w o u l d h a v e been m i n i m a l . R e s u l t s a re e x p r e s s e d as m e a n ± S E ; n , n u m b e r s o f a n i m a l s ; ^ s i g n i f i c a n t l y d i f f e r e n t f r o m u n t r e a t e d c o n t r o l s (p<0.05); t s i g n i f i c a n t l y d i f f e r e n t f r o m the o t h e r three groups (p<0.05). 6 4 pressures g r e a t e r t h a n 12.5 c m ^ O . A t f i l l i n g pressures less t h a n 12.5 c m H2O, the pressure d e v e l o p e d i n h y d r a l a z i n e - t r e a t e d d i a b e t i c s was s t i l l s i g n i f i c a n t l y l o w e r t h a n u n t r e a t e d c o n t r o l s . T h i s was also t r u e w h e n h y d r a l a z i n e - t r e a t e d c o n t r o l s w e r e c o m p a r e d to u n t r e a t e d c o n t r o l s . S i m i l a r r e s u l t s w e r e also seen for the r a t e o f pressure d e c l i n e ( - d P / d t ) . H o w e v e r i n th i s case , t h e r e w a s no d i f f e r e n c e b e t w e e n the u n t r e a t e d c o n t r o l and h y d r a l a z i n e - t r e a t e d c o n t r o l groups . F o r the l e f t v e n t r i c u l a r pressure d e v e l o p m e n t (+dP/dt) , h y d r a l a z i n e p r e t r e a t m e n t of the d i a b e t i c r a t s o n l y p a r t i a l l y r e v e r s e d th i s depressed p a r a m e t e r seen in u n t r e a t e d d i a b e t i c s . H e r e t o o , h y d r a l a z i n e - t r e a t e d c o n t r o l s had l o w e r +dP/dt v a l u e s , th is t i m e at f i l l i n g pressures g r e a t e r t h a n 17.5 c m w h e n c o m p a r e d to u n t r e a t e d c o n t r o l s . These r e s u l t s c o n f i r m the o b s e r v a t i o n that u n t r e a t e d d i a b e t i c r a t s are c h a r a c t e r i z e d by a depressed c a r d i a c p e r f o r m a n c e . In c o n t r a s t , the p e r f o r m a n c e o f the hear t s o f the h y d r a l a z i n e - t r e a t e d d i a b e t i c r a t s s h o w e d a d e f i n i t e i m p r o v e m e n t . T h e h e a r t f u n c t i o n o f the h y d r a l a -z i n e - t r e a t e d c o n t r o l s a lso a p p e a r e d to be a f f e c t e d . 4 . C H A N G E IN S E R U M L I P I D S A F T E R H Y D R A L A Z I N E : F i g u r e s 10-13 show the s e r u m l i p i d p r o f i l e in the e x p e r i m e n t a l r a t s . S e r u m f r e e f a t t y ac ids ( F i g . 10), t r i g l y c e r i d e s ( F i g . 11), t o t a l c h o l e s t e r o l ( F i g . 12) and phosphol ip ids ( F i g . 13) w e r e m a r k e d l y e l e v a t e d i n the u n -t r e a t e d d i a b e t i c g r o u p . In c o n t r a s t , h y d r a l a z i n e t r e a t m e n t o f the d i a b e t i c r a t s r e s t o r e d these p a r a m e t e r s to n o r m a l . T h e s e r u m l i p i d s o f the h y d r a -l a z i n e - t r e a t e d c o n t r o l s a p p e a r e d to be n o r m a l . H e n c e i t seems that h y d r a l a z i n e c o n t r o l l e d the e l e v a t e d s e r u m l i p i d s o f the d i a b e t i c r a t s h a v i n g no e f f e c t in the c o n t r o l s . 65 F I G U R E 9 E f f e c t of d i a b e t e s and h y d r a l a z i n e t r e a t m e n t on (A) l e f t v e n t r i c u l a r d e v e l o p e d pressure , (B) - d P / d t , and (C) +dP/dt of i s o l a t e d p e r f u s e d w o r k i n g h e a r t s at v a r i o u s f i l l i n g pressures . H e a r t s w e r e i s o l a t e d f r o m u n t r e a t e d c o n t r o l ( O - O , n=8), h y d r a l a z i n e - t r e a t e d c o n t r o l ( A - A , n=6), u n t r e a t e d d i a b e t i c ( • - • , n=8), and h y d r a l a z i n e - t r e a t e d d i a b e t i c ( • - • , n=8) r a t s 42 days a f t e r the i n d u c t i o n o f d i a b e t e s w i t h 55 m g / k g i . v . s t r e p t o z o t o c i n . R e s u l t is the m e a n ± S E for n u m b e r of h e a r t s s h o w n i n parentheses . * p 0.05 vs . u n t r e a t e d c o n t r o l . + p<0.05 vs . a l l o t h e r groups . + + p < 0 . 0 5 vs . h y d r a l a z i n e - t r e a t e d c o n t r o l . 67 F I G U R E 10 E f f e c t of d i a b e t e s and h y d r a l a z i n e t r e a t m e n t on s e r u m f r e e f a t t y ac ids of e x p e r i m e n t a l r a t s . R e s u l t is the m e a n ± S E f o r the n u m b e r of a n i m a l s shown i n parentheses . A s t e r i s k s i n d i c a t e s i g n i f i -c a n t d i f f e r e n c e s f r o m a l l o t h e r groups , p<0.05. 68 ^ 2 r LU |~| U N T R E A T E D C O N T R O L (n - 8) §§§ U N T R E A T E D DIABETIC (n - B) | H Y D R A L A Z I N E - T R E A T E D C O N T R O L (n « 6) flTI H Y D R A L A Z I N E - T R E A T E D DIABETIC (n - 8) 69 F I G U R E 11 E f f e c t o f d i a b e t e s and h y d r a l a z i n e t r e a t m e n t on s e r u m t r i g l y -c e r i d e s of e x p e r i m e n t a l r a t s . R e s u l t is the m e a n +SE for the n u m b e r o f a n i m a l s shown i n parentheses . A s t e r i s k s i n d i c a t e s i g n i f i c a n t d i f f e r e n c e s f r o m a l l o t h e r groups , p<0.05. 70 E 1 0 0 0 n U N T R E A T E D C O N T R O L (n • B) U N T R E A T E D OIABETIC (n - 8) H Y D R A L A Z I N E - T R E A T E D C O N T R O L (n - B) fTTl H Y D R A L A Z I N E - T R E A T E D DIABETIC (n « B) 71 F I G U R E 12 E f f e c t o f d i a b e t e s and h y d r a l a z i n e t r e a t m e n t on s e r u m t o t a l c h o l e s t e r o l o f e x p e r i m e n t a l r a t s . R e s u l t is the m e a n ± S E for the n u m b e r of a n i m a l s shown in parentheses . A s t e r i s k s i n d i c a t e s i g n i f i -c a n t d i f f e r e n c e s f r o m a l l o t h e r groups , p<0.05. (~1 U N T R E A T E D C O N T R O L (n « 8) S3 U N T R E A T E D DIABETIC (n = B) | H Y D R A L A Z I N E - T R E A T E D C O N T R O L (n = ATI H Y D R A L A Z I N E - T R E A T E D DIABETIC (n = 73 F I G U R E 13 E f f e c t o f d i a b e t e s and h y d r a l a z i n e t r e a t m e n t on s e r u m phospho-l i p i d s of e x p e r i m e n t a l r a t s . R e s u l t is the m e a n +SE for the n u m b e r of a n i m a l s shown in parentheses . A s t e r i s k s i n d i c a t e s i g n i f i c a n t d i f f e r e n c e s f r o m a l l o t h e r groups , p<0.05. 74 Oil H Y D R A L A Z I N E - T R E A T E D DIABETIC (n - 8) 75 F I G U R E 14 D o s e - r e s p o n s e c u r v e s of h y d r a l a z i n e on t e n s i o n and r a t e d e v e l -o p m e n t o f i s o l a t e d l e f t and r i g h t a t r i a o b t a i n e d f r o m 6 w e e k c o n t r o l ( • ) and S T Z ( O / - t r e a t e d r a t s . E a c h po in t r e p r e s e n t s the m e a n ± S E for the n u m b e r of a n i m a l s s h o w n in parentheses . D a t a are e x p r e s s e d as a b s o l u t e v a l u e s (y-axis) p r o d u c e d by v a r i o u s m o l a r c o n c e n t r a t i o n s of h y d r a l a z i n e (x -ax is ) . 76 77 5. E F F E C T O F H Y D R A L A Z I N E O N R I G H T A N D L E F T A T R I A : H y d r a l a z i n e p r o d u c e d a dose -dependent d e c r e a s e in r i g h t a t r i a l r a t e o f c o n t r o l as w e l l as d i a b e t i c r a t s . H y d r a l a z i n e a lso p r o d u c e d a p o s i t i v e _3 i n o t r o p i c e f f e c t in the l e f t a t r i u m at a v e r y h igh dose (1x10 and _3 3x10 M ) . T h i s response was i d e n t i c a l i n c o n t r o l as w e l l as d i a b e t i c p r e p -a r a t i o n s ( F i g u r e 14). D . M A L E A N D F E M A L E S T U D Y : I n j e c t i n g a n i m a l s w i t h S T Z r e s u l t e d i n a d i a b e t i c s t a t e w i t h an e l e v a t -e d u r i n e g l u c o s e (>2%) t h r o u g h o u t the s tudy p e r i o d . O t h e r s y m p t o m s n o r m a l l y a s s o c i a t e d w i t h the d i a b e t i c s t a t e such as p o l y u r i a , p o l y d i p s i a and p o l y p h a g i a w e r e seen i n the d i a b e t i c r a t s . M a l e d i a b e t i c r a t s demons-t r a t e d a 13% g r e a t e r loss i n body w e i g h t w h e n c o m p a r e d to f e m a l e d i a b e t -i c r a t s . O n l y in the case of m a l e d i a b e t i c r a t s was t h e r e a s i g n i f i c a n t d e c r e a s e i n d r y hear t w e i g h t and an i n c r e a s e in d r y hear t w e i g h t to body w e i g h t r a t i o w h e n c o m p a r e d to m a l e c o n t r o l s . S e r u m g l u c o s e , c h o l e s t e r o l and t r i g l y c e r i d e s w e r e m a r k e d l y e l e v a t e d in b o t h d i a b e t i c groups w h e r e a s s e r u m T - 4 and i n s u l i n l e v e l s w e r e s i g n i f i c a n t l y l o w e r w h e n c o m p a r e d w i t h the r e s p e c t i v e c o n t r o l s . H o w e v e r , m a l e d i a b e t i c ra ts d e m o n s t r a t e d a 2 5 % g r e a t e r drop i n s e r u m i n s u l i n w h e n c o m p a r e d to f e m a l e d i a b e t i c r a t s . T h e s e r e s u l t s are s u m m a r i z e d i n T a b l e 9. F i g u r e 15 i n d i c a t e s the e f f e c t of v a r y i n g f i l l i n g pressures on r a t e o f pressure d e v e l o p m e n t (+dP/dt) in c o n t r o l and d i a b e t i c r a t s . C o n t r a c t i l i t y (+dP/dt) was depressed in f e m a l e d i a b e t i c ra ts but o n l y at l e f t a t r i a l f i l l i n g pressures g r e a t e r than 17.5 c m ^ 0 . H o w e v e r ^ in m a l e d i a b e t i c r a t s , +dP/dt was depressed at a l l l e f t a t r i a l f i l l i n g pressures . W h e n m a l e T A B L E 9. V A R I O U S P A R A M E T E R S U S E D T O A S S E S S T H E D I A B E T I C S T A T E O F E X P E R I M E N T A L R A T S (male and f e m a l e s tudy) C O N T R O L S T R E P T O Z O T O C I N - I N J E C T E D F e m a l e n=9 M a l e n=8 F e m a l e n=10 M a l e n=9 B o d y w e i g h t (g) 2 2 3 ± 3 3 7 3 ± 7 1 9 5 ± 5 a * 2 7 9 ± 3 b * D r y heart w e i g h t (mg) 108+4 165±4 1 0 0 ± 4 a N S 1 4 5 ± 3 b * D r y heart w e i g h t / j body weight (mg.g ) 0 . 4 8 5 ± 0 . 0 2 0 . 4 4 8 ± 0 . 0 1 0 . 5 1 7 ± 0 . 0 2 a N S 0 . 5 1 8 ± 0 . 0 1 b * S e r u m glucose (mg %) 142±12 145±7 6 3 9 ± 3 2 a * 5 7 4 ± 3 5 b * S e r u m i n s u l i n ( y U / m L ) 3 2 ± 4 7 2 ± 8 15+3 a * 1 6 ± 5 b * S e r u m T M g / 1 0 0 m L ) 5 . 1 7 ± 0 . 4 1 5 . 3 4 ± 0 . 4 3 . 1 6 ± 0 . 2 6 a * 2 . 8 3 ± 0 . 2 3 b * S e r u m c h o l e s t e r o l (mg/100 m L ) 5 6 ± 5 4 8 ± 3 9 9 ± 1 5 a * 1 0 0 ± 1 0 b * S e r u m t r i g l y c e r i d e (mg/100 m L ) 9 5 ± 8 113±5 6 4 4 ± 9 8 a * 6 7 0 ± 1 3 8 b * R e s u l t s are expressed as m e a n + S . E . M . ; n , n u m b e r of a n i m a l s ; C o m p a r e d to f e m a l e c o n t r o l ; c o m p a r e d to m a l e c o n t r o l ; *p<0.05; N S , not s i g n i f i c a n t . 79 F I G U R E 15 E f f e c t o f d i a b e t e s on M-dP/dt i n (A) f e m a l e , and (B) m a l e i s o l a -t e d p e r f u s e d w o r k i n g hear t s at v a r i o u s f i l l i n g pressures . H e a r t s w e r e i s o l a t e d f r o m f e m a l e c o n t r o l ( O - O , n=9), f e m a l e d i a b e t i c ( > - > , n=10), m a l e c o n t r o l ( • - # , n=8) and m a l e d i a b e t i c (• - • , n=9) r a t s 42 days a f t e r the i n d u c t i o n of d i a b e t e s w i t h 55 m g / k g i . v . s t r e p t o z o t o c i n . R e s u l t is the m e a n +SE f o r the n u m b e r o f h e a r t s shown i n parentheses . * p 0.05 vs . c o n t r o l o f the s a m e sex . + p<0.05 vs . f e m a l e d i a b e t i c s . 81 diabetics were compared to female diabetics, there was a lower pressure development in male diabetics at most filling pressures. Similar results were also seen for the LVDP and -dP/dt. Cardiac function in the male diabetic rats was the lowest of the four groups. 82 D I S C U S S I O N T h e resu l t s p r e s e n t e d here suppor t the e a r l i e r f i n d i n g s o f F a c t o r et a l . ( F a c t o r et a l . , 1980; F a c t o r et a l . , 1981; F a c t o r et a l . , 1983; F e i n et a l . , 1984) in the r e n o v a s c u l a r h y p e r t e n s i v e - d i a b e t i c r a t m o d e l tha t the c o m b i n a t i o n of d iabe tes m e l l i t u s and s y s t o l i c h y p e r t e n s i o n p r o d u c e s p r o f o u n d c a r d i o v a s c u l a r changes i n the r a t . T h e degree of these changes v a r i e s w i t h the d u r a t i o n o f the h y p e r t e n s i v e - d i a b e t i c s t a t e , i . e . 6 w e e k s f o l l o w i n g S T Z i n j e c t i o n no d i f f e r e n c e s i n c a r d i a c f u n c t i o n w e r e o b s e r v e d b e t w e e n the n o r m o t e n s i v e and h y p e r t e n s i v e d i a b e t i c group, w h e r e a s at 12 w e e k s t h e r e was a s i g n i f i c a n t d e p r e s s i o n i n c a r d i a c f u n c t i o n i n the h y p e r t e n s i v e - d i a b e t i c group a s s o c i a t e d w i t h an i n c r e a s e d m o r t a l i t y . In our 6 w e e k S H R s t u d y W i s t a r r a t s w e r e used as n o r m o t e n s i v e c o n t r o l s . T r e a t m e n t w i t h S T Z r e s u l t e d i n a d i a b e t i c s t a t e c h a r a c t e r i z e d by h y p e r g l y c e m i a and loss o f body w e i g h t as e a r l y as 3 days a f t e r t r e a t -m e n t . T h e d e c r e a s e in body and hear t w e i g h t at the t i m e o f s a c r i f i c e o c c u r r e d i n s p i t e o f i n c r e a s e d w a t e r and f o o d i n t a k e ( H o f t e i z e r and C a r p e n t e r , 1973) and was p r o b a b l y due to d e h y d r a t i o n and to p r o t e i n and f a t c a t a b o l i s m seen d u r i n g d i a b e t e s ( O a k l e y , 1968). T h e r e s u l t s r e g a r d i n g the e f f e c t s of S T Z d i a b e t e s on b l o o d pressure and h e a r t a re c o n s i s t e n t w i t h p r e v i o u s r e p o r t s . N o r m o t e n s i v e W i s t a r r a t s m a d e d i a b e t i c d e v e l o p e d a s m a l l i n c r e a s e i n b l o o d pressure , i n a g r e e m e n t w i t h the r e s u l t s o f B u n a g et a l . (Bunag et a l . , 1982) who a lso r e p o r t e d t h a t a l t h o u g h d i a b e t i c r a t s are p r e d i s p o s e d to b e c o m e h y p e r t e n s i v e , o t h e r m e c h a n i s m s s u c h as h y p o t h a l a m i c depress ion m a y be a c t i v a t e d to r e s t r i c t f u r t h e r e l e v a t i o n in b l o o d p r e s s u r e . In a l a t e r r e p o r t t h e y s h o w e d t h a t d a i l y subcutaneous i n j e c t i o n of L e n t e i n s u l i n r e d u c e d the h y p e r t e n s i o n w h i c h 83 d e v e l o p e d c o n s i s t e n t l y in t h e i r r a t s (Sasaki and B u n a g , 1983). In our s t u d y , b l o o d p r e s s u r e p r o g r e s s i v e l y i n c r e a s e d and r e a c h e d i t s m a x i m u m response s ix w e e k s a f t e r the a d m i n i s t r a t i o n of S T Z . What m a k e s d i a b e t i c r a t s s u s c e p t i b l e to h y p e r t e n s i o n r e m a i n s u n c e r t a i n . D e s p i t e i n c r e a s e s i n c a r d i o v a s c u l a r r e a c t i v i t y w h i c h have been r e p o r t e d i n d i a b e t i c p a t i e n t s ( W e i d m a n n et a l . , 1979) and a l l o x a n d i a b e t i c r a t s (Brody and D i x o n , 1964), B u n a g et a l . (1982) f o u n d s y s t e m i c pressor responses to i n j e c t e d n o r -e p i n e p h r i n e u n a l t e r e d in the S T Z d i a b e t i c r a t . S i m i l a r l y , the r e n i n - a n g i o -t e n s i n s y s t e m is suppressed i n a l l o x a n d i a b e t i c ra ts t h a t b e c o m e h y p e r -t e n s i v e ( C h r i s t l i e b , 1974). A n e x p l a n a t i o n f o r the i n c r e a s e i n b l o o d p r e s s u r e m a y be r e n a l d isease , w h i c h is c o m m o n i n c l i n i c a l d i a b e t e s ( C h r i s t l i e b , 1973) and in r a t s t r e a t e d w i t h S T Z ( R a s c h , 1980). T h e l a c k o f e f f e c t o f d i a b e t e s on S H R b l o o d pressure at 6 w e e k s is not c o n s i s t e n t w i t h the r e s u l t s of H a s h i m o t o ( H a s h i m o t o , 1969) who r e p o r t e d a h y p e r t e n -s i v e e f f e c t . T h e reasons f o r the d i s p a r i t y b e t w e e n r e s u l t s m a y be due to the longer d u r a t i o n o f H a s h i m o t o ' s e x p e r i m e n t ( d i a b e t i c up to 18 months) and the f a c t tha t he used a l l o x a n to i n d u c e d i a b e t e s . F u r t h e r -m o r e , the a d d i t i o n o f sa l t to his a n i m a l s ' f e e d m a y not o n l y have m a i n t a i n e d f l u i d v o l u m e , but also e l e v a t e d b l o o d p r e s s u r e . O u r r e s u l t s r e g a r d i n g the e f f e c t s o f s t r e p t o z o t o c i n - d i a b e t e s on hear t r a t e are also c o n s i s t e n t w i t h p r e v i o u s r e p o r t s . B r a d y c a r d i a o c c u r s w h e n d i a b e t e s is i n d u c e d i n n o r m o t e n s i v e r a t s w i t h s t r e p t o z o t o c i n ( J a c k s o n and C a r r i e r , 1983a), or in s p o n t a n e o u s l y h y p e r t e n s i v e ones w i t h a l l o x a n ( C a v a l i e r e et a l . , 1980). In the 6 w e e k s t u d y , h e a r t r a t e s o f D I A and S H D r a t s w e r e r e d u c e d by the f i r s t w e e k a f t e r S T Z i n j e c t i o n . T h e u n d e r -l y i n g m e c h a n i s m s f o r th is b r a d y c a r d i a are o b s c u r e . S a v a r e s e and B e r k o w i t z 84 h a v e s p e c u l a t e d that the d e c r e a s e i n h e a r t r a t e m a y be due to a d e c r e a s e i n the n u m b e r of b e t a - a d r e n o c e p t o r b i n d i n g s i tes t h a t they o b s e r v e d i n the d i a b e t i c hear t (Savarese and B e r k o w i t z , 1979). T h e y r e p o r t e d a 28% r e d u c t i o n in the b e t a r e c e p t o r s a c c o m p a n i e d by a 24% d e c r e a s e i n hear t r a t e f o r 8 - w e e k S T Z - i n j e c t e d d i a b e t i c a n i m a l s w h e n c o m p a r e d w i t h c o n t r o l s . T h e y a t t r i b u t e d the d e c r e a s e in b e t a - r e c e p t o r s to the l o w l e v e l o f t h y r o i d h o r m o n e in the a f f e c t e d a n i m a l s , h y p o t h y r o i d i s m b e i n g k n o w n to cause a r e d u c t i o n in the d e n s i t y of b o t h a l p h a - and b e t a - r e c e p t o r s i n c a r d i a c t i ssue ( C i a r a l d i and M a r i n e t t i , 1977). O t h e r authors h a v e a lso s h o w n t h a t the i n d u c t i o n o f d i a b e t e s by S T Z and a l l o x a n w i l l p r o d u c e a h y p o t h y r o i d s t a t e i n e x p e r i m e n t a l a n i m a l s ( F e i n et a l . , 1980; V a d l a m u d i and M c N e i l l , 1983; G a r b e r et a l . , 1983). A d d i t i o n a l e v i d e n c e f o r the i n v o l v e m e n t of t h y r o i d h o r m o n e s i n the d e c r e a s e in h e a r t r a t e is tha t T g t r e a t m e n t of S T Z d i a b e t i c r a t s p r e v e n t e d this b r a d y c a r d i a ( G o y a l and M c N e i l l , 1985), W i s t a r - K y o t o r a t s m a d e d i a b e t i c w i t h S T Z f a i l e d to show the c h a r a c t e r i s t i c d e c r e a s e in h e a r t r a t e and w e r e subsequent ly f o u n d to have n o r m a l l e v e l s of t h y r o i d h o r m o n e s ( R o d r i g u e s et a l . , 1985) and T ^ and T ^ l e v e l s w e r e r e d u c e d as e a r l y as 8 days a f t e r s a c r i f i c e of S T Z d i a b e t i c r a t s (GeStzche, 1983). In a d d i t i o n , t h y r o i d h o r m o n e s have d i r e c t e f f e c t s on hear t r a t e and c o n t r a c t i l i t y m e c h a n i s m s independent o f c a t e c h o l a m i n e s . L o s s of b e t a - a d r e n e r g i c r e c e p t o r s m a y also be due to a ' d o w n - r e g u l a t i o n ' m e c h a n i s m , s i n c e e l e v a t e d p l a s m a c a t e c h o l a m i n e l e v e l s have been r e p o r t e d i n e x p e r i m e n t a l and c l i n i c a l d iabe tes (Paulson et a l . , 1980; C h r i s t e n s e n , 1974). H i g h c a t e c h o l a m i n e l e v e l s are k n o w n to r e d u c e b e t a r e c e p t o r d e n s i t y ( M i c k e y et a l . , 1975). H e a r t s f r o m 6 w e e k D I A and S H D r a t s e x h i b i t e d a d e p r e s s i o n i n 85 c a r d i a c f u n c t i o n c o m p a r e d to t h e i r c o n t r o l s . W h e n w e c o m p a r e d the c a r d i a c f u n c t i o n o f D I A and S H D r a t s to e a c h o t h e r , w e f o u n d that the f u n c t i o n was e q u i v a l e n t l y depressed i n the t w o groups , sugges t ing that h y p e r t e n s i o n does not enhance the c a r d i a c f u n c t i o n a l a l t e r a t i o n s seen d u r i n g d i a b e t e s at th is t i m e p e r i o d . P r e v i o u s r e p o r t s by F a c t o r ' s group ( F e i n et aV;-j 1984) have been w i t h r e n o v a s c u l a r h y p e r t e n s i v e - d i a b e t i c r a t s of 8 w e e k s d u r a t i o n , so i t was y e t to be seen w h a t w o u l d happen i f the d u r a t i o n of our h y p e r t e n s i v e - d i a b e t i c s t a t e was i n c r e a s e d . F o r the 12 w e e k p e r i o d o f c o m b i n e d d i a b e t e s and h y p e r t e n s i o n , the W i s t a r - K y o t o r a t , c o n s i d e r e d to be the best n o r m o t e n s i v e c o n t r o l f o r the S H R a n i m a l s (S joerdsme, 1972), was a lso i n c l u d e d . S e r u m g lucos e and g l y c o s y l a t e d h e m o g l o b i n l e v e l s w e r e f o u n d to be c o n s i s t e n t l y h i g h i n a l l the r a t s r e c e i v i n g the d i a b e t o g e n w i t h c o r r e s p o n d i n g h y p o i n s u l i n e m i a . S H D , W K D , and D I A r a t s e x h i b i t e d u r i n a r y g lucose l e v e l s g r e a t e r t h a n 2% t h r o u g h o u t the s t u d y . H o w e v e r , e v e n at s i m i l a r degrees o f h y p e r -g l y c e m i a and h y p o i n s u l i n e m i a , w e i g h t loss in the W K D r a t s , as c o m p a r e d to the o t h e r t w o d i a b e t i c groups , was not as g r e a t . T h e d r y h e a r t w e i g h t - t o - b o d y w e i g h t r a t i o was e l e v a t e d o n l y in S H D and D I A r a t s , w i t h S H D r a t s h a v i n g the g r e a t e s t degree o f m y o c a r d i a l h y p e r t r o p h y . A l t h o u g h an u l t r a s t r u c t u r a l a n a l y s i s o f the m y o c a r d i u m w a s not done i n th is i n v e s t i g a t i o n , m o r p h o l o g i c a l s tudies o f h e a r t s f r o m d i a b e t i c r a t s have sugges ted these h e a r t s to be h y p e r t r o p h i c at the c e l l u l a r l e v e l (Onish i et a l . , 1981). T h e r e w a s a t e n d e n c y t o w a r d s e l e v a t e d s y s t o l i c b l o o d pressure i n the d i a b e t i c W i s t a r r a t as seen in the 6 w e e k s t u d y . In c o m -p a r i s o n , S H D r a t s e x h i b i t e d a r e d u c t i o n i n s y s t o l i c pressure as c o m p a r e d to S H C r a t s by the 9 th w e e k . T h e h y p o t e n s i v e e f f e c t of S T Z - i n d u c e d 86 d i a b e t e s on S H D r a t s is i n a g r e e m e n t w i t h p r e v i o u s authors ( C a v a l i e r et a l . , 1980; S o m a n i et a l . , 1979), l a m s and W e x l e r (1977) a t t r i b u t i n g the f a l l in b l o o d pressure up to 8 w e e k s a f t e r a d m i n i s t r a t i o n o f a l l o x a n p r i m a r i l y to i m p a i r e d a d r e n o c o r t i c a l s t e r o i d o g e n e s i s . D e p e n d i n g on w h i c h S H D c o l o n y is s t u d i e d , and perhaps on the i n b r e e d i n g t e c h n i q u e s f o l l o w e d , a r t e r i a l pressures u s u a l l y are e l e v a t e d f r o m a v e r y e a r l y age i n the S H R and u s u a l l y i n c r e a s e w i t h age, r e a c h i n g a m a x i m u m at a p p r o x i m a t e l y 20-28 w e e k s ( O k a m o t o et a l . , 1972). T h e p r e c i s e p a t h o p h y s i o l o g i c a l m e c h -an isms respons ib le f o r the p r o d u c t i o n and m a i n t e n a n c e of h y p e r t e n s i o n in the S H R are not f u l l y u n d e r s t o o d . T h e r e is some e v i d e n c e o f i n c r e a s e d s y m p a t h o a d r e n a l a c t i v i t y and i n c r e a s e d v a s c u l a r respons iveness to c a t e c h o l -a m i n e s d u r i n g the d e v e l o p m e n t of h y p e r t e n s i o n in S H R ( D i e t z et a l . , 1978). In our s tudy , a l t h o u g h the b l o o d pressure in the S H D a n i m a l s was l o w e r t h a n that of the S H C r a t a f t e r 9 w e e k s , i t a p p e a r e d to be due to the f a c t t h a t the p r o g r e s s i v e r i se in b l o o d pressure that is seen in the S H C r a t is c u r t a i l e d in the S H D a n i m a l . In a d d i t i o n , Y o u n g et a l . (1977) h a v e r e p o r t e d t h a t c a l o r i c r e s t r i c t i o n l o w e r s s y m p a t h e t i c a c t i v i t y i n n o r m a l r a t s and l o w e r s b l o o d pressure in S H R . H e n c e i t is not c l e a r w h e t h e r the c a l o r i c d e p r i v a t i o n seen i n these r a t s or the d i a b e t i c s t a t e per se c o u l d a c c o u n t f o r the h y p o t e n s i v e e f f e c t s . In the 12 w e e k s tud y , h e a r t r a t e s o f S H D and D I A r a t s w e r e r e d u c e d s i g n i f i c a n t l y i n c o m p a r i s o n w i t h age m a t c h e d c o n t r o l s . H o w e v e r , W K D r a t s d i d not show the c h a r a c -t e r i s t i c d e c r e a s e i n hear t r a t e seen i n the o t h e r d i a b e t i c groups , i n a g r e e -m e n t w i t h a p r e v i o u s r e p o r t us ing a l l o x a n d i a b e t i c W K Y r a t s ( C a v a l i e r et a l . , 1980). A n a s s o c i a t i o n b e t w e e n h y p e r l i p i d e m i a and d i a b e t i c k e t o a c i d o s i s 87 w a s f i r s t d e m o n s t r a t e d by B l o o r (1916). S i n c e t h e n , b l o o d l i p i d s have f a i r l y c o m m o n l y been r e p o r t e d to be e l e v a t e d a m o n g a m b u l a n t d i a b e t i c p a t i e n t s . T h e most c o m m o n l y e l e v a t e d f a s t i n g p l a s m a l i p i d l e v e l is t r i g l y -c e r i d e ( N i k k i l a , 1974) but the p l a s m a c h o l e s t e r o l c o n c e n t r a t i o n is a lso i n c r e a s e d in the d i a b e t i c p o p u l a t i o n ( A l b r i n k et a l . , 1963). E l e v a t i o n o f s e r u m l i p i d s i n d i c a t e s e i t h e r the d e f e c t i v e r e m o v a l or o v e r p r o d u c t i o n (or both) o f one or m o r e l i p o p r o t e i n s ( F i g . 16). S i n c e i n s u l i n p l a y s a r o l e i n b o t h p r o d u c t i o n and r e m o v a l o f t r i g l y c e r i d e - r i c h l i p o p r o t e i n s , i t m a y be the m a j o r cause o f l i p i d d i s o r d e r s of d i a b e t e s . T h e r e is s t r o n g e v i d e n c e f o r a d e f e c t in the r e m o v a l of t r i g l y c e r i d e - r i c h l i p o p r o t e i n i n i n s u l i n d e f i c i e n t a n i m a l s and m a n t h a t is r e s t o r e d to n o r m a l by a d m i n i s -t r a t i o n o f i n s u l i n ( B r u n z e l l et a l . , 1979). S i n c e l i p o p r o t e i n l ipase ( L P L ) is r e s p o n s i b l e f o r the r e m o v a l of the t r i g l y c e r i d e - r i c h l i p o p r o t e i n s , a t t e n t i o n has f o c u s e d on th is e n z y m e w h i c h r e q u i r e s the p r e s e n c e o f i n s u l i n f o r m a i n t e n a n c e of adequate t i ssue l e v e l s and a c t i v i t y . Indeed, L P L a c t i v i t y has been d e m o n s t r a t e d to f a l l a f t e r i n s u l i n w i t h d r a w a l i n i n s u l i n - d e p e n d e n t d i a b e t i c a n i m a l s and m a n and is l o w in p o o r l y c o n t r o l l e d d i a b e t i c sub jec ts ( H a v e l , 1976). T h e d a t a for s e r u m c h o l e s t e r o l and t r i g l y -c e r i d e i n the D I A and S H D r a t d e m o n s t r a t e the r e l a t i o n s h i p b e t w e e n e l e v a t e d l e v e l s o f these l i p i d s and l o w s e r u m i n s u l i n l e v e l s seen i n these a n i m a l s . U n e x p e c t e d l y , W K D r a t s , a l t h o u g h they had l o w s e r u m i n s u l i n , d i d not have the e l e v a t e d s e r u m c h o l e s t e r o l and t r i g l y c e r i d e s seen i n the o t h e r d i a b e t i c groups . A n o t h e r b a s i c b i o c h e m i c a l d i s t u r b a n c e s i n i n s u l i n d e f i c i e n c y is a m a r k e d i n c r e a s e in adipose t issue l i p o l y s i s a c c o m p a n i e d by e n h a n c e d r e l e a s e of f r e e f a t t y ac ids ( M c G a r r y and F o s t e r , 1977). In n o r m a l l i v e r , an i n -88 c r e a s e d a m o u n t of f ree f a t t y ac ids p r o v i d e s a m a j o r s u b s t r a t e supply f o r h e p a t i c l ipogenes is and c a n be s e c r e t e d and r e c i r c u l a t e d on p l a s m a v e r y l o w d e n s i t y l i p o p r o t e i n - t r i g l y c e r i d e . W h e t h e r a s i m i l a r r e l a t i o n s h i p e x i s t s in i n s u l i n depressed l i v e r is d i f f i c u l t to d e c i d e because p u b l i s h e d d a t a are c o n t r o v e r s i a l and both depressed and i n c r e a s e d p r o d u c t i o n of t r i g l y c e r i d e and v e r y l o w d e n s i t y l i p o p r o t e i n ( V L D L ) has been r e p o r t e d . N e v e r t h e l e s s , i t appears that the s e c r e t i o n o f V L D L i n t o p l a s m a m a y be r e d u c e d w h e n i n s u l i n is v i r t u a l l y absent , w h e r e a s i n c r e a s e d V L D L and t r i g l y c e r i d e p r o d u c t i o n i n r a t s has been r e p o r t e d w i t h less s e v e r e i n s u l i n d e f i c i e n c y ( N i k k i l a , 1984). T r i g l y c e r i d e l ipase is the r a t e - d e t e r m i n i n g e n z y m e in adipose t issue l i p o l y s i s ( F i g . 16). It is an i n s u l i n - i n h i b i t e d , c y c l i c A M P - d e p e n d e n t e n z y m e w h i c h , under the i n f l u e n c e o f e p i n e p h r i n e , g l u c a g o n and v a r i o u s o t h e r h o r m o n e s , h y d r o l y z e s t r i g l y c e r i d e i n t o d i g l y c e r i d e s , u n e s t e r i f i e d f a t t y ac ids and f r e e g l y c e r o l . T h e f a t t y ac ids thus p r o d u c e d m a y d i f f u s e out o f the c e l l or t h e y m a y be r e e s t e r i f i e d i n t o t r i g l y c e r i d e w i t h i n the a d i p o c y t e . T h e r e e s t e r i f i c a t i o n process , h o w -e v e r , r e q u i r e s a l p h a - g l y c e r o p h o s p h a t e w h i c h is p r o v i d e d by i n s u l i n p r o m o t i n g the f l u x of g l u c o s e i n t r a c e l l u l a r l y . In the s e t t i n g o f i n s u l i n l a c k , t r i g l y -c e r i d e l i p a s e ac t s w i t h o u t o p p o s i t i o n and f r e e f a t t y a c i d s as w e l l as g l y c e r o l a re r e l e a s e d in l a r g e q u a n t i t i e s f r o m the adipose t i ssue (Saudek and E d e r , 1979). O u r r e s u l t s r e g a r d i n g the e f f e c t s of S T Z d i a b e t e s on s e r u m f r e e f a t t y ac ids i n S H D and D I A r a t s are c o n s i s t e n t w i t h those of N i k k i l a (1984). H o w e v e r , W K D r a t s f a i l e d to show the e l e v a t e d f r e e f a t t y ac ids seen i n the o t h e r t w o d i a b e t i c groups . T h i s c o u l d p a r t i a l l y e x p l a i n the l a c k of e l e v a t i o n i n s e r u m c h o l e s t e r o l and t r i g l y c e r i d e that is o b s e r v e d in these r a t s . W h y s e r u m l i p i d l e v e l s are not e l e v a t e d i n 89 W K D r a t s e v e n in a s e t t i n g of i n s u l i n l a c k of u n k n o w n . G o l d b e r g (1981) has sugges ted that the a m o u n t o f f a t t y ac ids h y d r o l y z e d f r o m l i p o p r o t e i n s , d e p o s i t e d f r o m t issue s tores , or r e l e a s e d f r o m the l a t t e r is n o r m a l l y an i n t e g r a t e d process , f i n e l y t u n e d by the a c t i o n o f i n s u l i n and the c o u n t e r r e g u l a t o r y hormones, g l u c a g o n , Cortisol and the catecholamines, with i n s u l i n p l a y i n g a m a j o r r o l e i n th is r e g u l a t i o n . T h e m e t a b o l i c changes o b s e r v e d in u n t r e a t e d d i a b e t e s a re , i n m a n y r e s p e c t s , s i m i l a r to those p r o d u c e d by i n f u s i o n o f c a t e c h o l a m i n e s and i n c l u d e h y p e r g l y c e m i a , d e c r e a s e d g l u c o s e t o l e r a n c e , e l e v a t e d f r e e f a t t y ac ids and k e t o n e bodies i n the p l a s m a . C a t e c h o l a m i n e s a re the most i m p o r t a n t h o r m o n e s a c u t e l y s t i m u l a t i n g the d e g r a d a t i o n o f the s t o r e d t r i g l y c e r i d e ( F i g . 16). T h e y b i n d to b e t a r e c e p t o r s of the f a t c e l l s and i n c r e a s e c A M P w h i c h i n t u r n leads to p h o s p h o r y l a t i o n and a c t i v a t i o n o f t r i g l y c e r i d e l i p a s e , r e s u l t i n g i n the d e g r a d a t i o n o f the s t o r e d t r i g l y c e r i d e to f a t t y a c i d and g l y c e r o l ( W i l l i a m - O T s s o n et a l . , 1979). D e c r e a s e d p l a s m a c o n c e n t r a t i o n s o f c a t e c h o -l a m i n e s have been r e p o r t e d i n h y p e r t h y r o i d i s m and r a i s e d c o n c e n t r a t i o n s i n h y p o t h y r o i d i s m as c o m p a r e d to the e u t h y r o i d s t a t e ( C h r i s t e n s e n , 1973). E l e v a t e d p l a s m a c a t e c h o l a m i n e l e v e l s h a v e also been r e p o r t e d i n e x p e r i -m e n t a l and c l i n i c a l d i a b e t e s ( C h r i s t e n s e n , 1974; P a u l s o n et a l . , 1980). Thus , a poss ib le e x p l a n a t i o n f o r the f a i l u r e in the W K D r a t s to show e l e v a t e d s e r u m l i p i d s c o u l d be that one of the c o u n t e r r e g u l a t o r y h o r m o n e s , p r e s u m a b l y c a t e c h o l a m i n e s , w h i c h are e l e v a t e d i n d i a b e t e s , w e r e not as e l e v a t e d i n these a n i m a l s w h i c h had n o r m a l c i r c u l a t i n g l e v e l s o f t h y r o i d h o r m o n e s . T h i s l a c k of e l e v a t i o n o f s e r u m l i p i d s i n these a n i m a l s c o u l d be another e x p l a n a t i o n f o r f a i l u r e in these a n i m a l s to show the c h a r a c t e r i s t i c drop i n hear t r a t e as e l e v a t e d p l a s m a l i p i d s have the 90 F I G U R E 16 L i p o p r o t e i n p a t h w a y s and m a j o r s teps i n the r e g u l a t i o n of t r i g l y c e r i d e m e t a b o l i s m in the a d i p o c y t e . B o t h t r i g l y c e r i d e - r i c h l i p o p r o t e i n s , n a m e l y c h y l o m i c r o n s and V L D L , undergo t r i g l y c e r i d e h y d r o l y s i s by l i p o p r o t e i n l i p a s e , r e s u l t i n g in the f o r m a t i o n of s m a l l e r t r i g l y c e r i d e - r i c h l i p o p r o t e i n s . F r e e f a t t y a c i d s , r e l e a s e d by the a c t i o n of l i p o p r o t e i n l ipase on t r i g l y c e r i d e - r i c h l i p o p r o t e i n s or f r o m adipose t i s sue s tores , a re t a k e n up by m a n y t i ssues , i n c l u d i n g adipose t i ssue , m u s c l e and l i v e r . S teps at w h i c h i n s u l i n e x e r t s a p o s i t i v e e f f e c t are m a r k e d (+); s teps w h i c h i n s u l i n i n h i b i t s are m a r k e d (-). L P L is l i p o p r o t e i n l ipase ; F F A is f r e e f a t t y a c i d ; G - 6 - P is g l y c e r o - 6 - p h o s p h a t e . ^ FREE FATTY ACIDS LIVER LIPOGENESIS CHOLESTEROL-TRIGLYCERIDE y A l e v / -ADIPOSE TISSUE + SKELETAL MUSCLE CHOLESTEROL + BILE ACIDS GUT ©TRIGLYCERIDE LIPASE / ADENYLATE CYCLASE" • c A M P ^ PROTEIN/ KINASE y , N A C T | V E TRIGLYCERIDE _ LIPASE INACTIVE PROTEIN KINASE FATTY ACID GLYCEROLS TRIGYCERIDES I -GP ' \ G - e - p DIETARY FAT © GLUCOSE 92 c a p a c i t y to a f f e c t b e t a - r e c e p t o r d e n s i t y in the hear t (Wince and R u t l e d g e , 1981). T h e t h y r o i d s ta tus of an a n i m a l is a lso k n o w n to a f f e c t the i n t r i n s i c c o n t r a c t i l e c a p a b i l i t i e s o f the h e a r t . H y p o t h y r o i d i s m has been s h o w n to s l o w r e l a x a t i o n and depress the f o r c e - v e l o c i t y r e l a x a t i o n i n p a p i l l a r y m u s c l e s ( B u c c i n o e t a l . , 1967), depress c a l c i u m t r a n s p o r t by the s a r c o p l a s -m i c r e t i c u l u m (Suko, 1971) and a c t o m y o s i n C a 2 + - A T P a s e a c t i v i t i e s (Baner jee , 1983) and s h i f t the m y o s i n i s o z y m e s u b t y p e d i s t r i b u t i o n s f r o m the " f a s t " V j f o r m to the " s l o w " V 3 f o r m (Baner jee , 1983). D i a b e t e s is k n o w n to have a n u m b e r of c a r d i a c , m e c h a n i c a l and b i o c h e m i c a l e f f e c t s s i m i l a r to those seen in h y p o t h y r o i d i s m . S p e c i f i c a l l y , i t r e d u c e s l e f t v e n t r i c u l a r c o n t r a c t i l i t y and r e l a x a t i o n r a t e ( V a d l a m u d i et a l . , 1982), c a l c i u m b i n d i n g and u p t a k e by the S R ( P e n p a r g k u l et a l . , 1980), m y o s i n and a c t o m y o s i n C a 2 + - A T P a s e a c t i v i t i e s ( M a l h o t r a , et a l . , 1981) and s h i f t s the p r e d o m i n a n t m y o s i n i s o z y m e s u b t y p e f r o m V j to the less a c t i v e ( M a l h o t r a et a l . , 1981). D i a b e t e s has a lso been shown to i n d u c e h y p o -t h y r o i d i s m i n v a r i o u s spec ies such as m a n and r a t and r e d u c e d l e v e l s o f p l a s m a T ^ or t h y r o x i n e have been r e p o r t e d in th is d isease ( D i l l m a n , 1982; G a r b e r et a l . , 1983). It is thus poss ib le tha t d i a b e t e s i n d u c e d f u n c -t i o n a l a l t e r a t i o n s c o u l d be a r e s u l t o f the h y p o t h y r o i d i s m w h i c h is seen i n d i a b e t e s . A s s h o w n in th is s t u d y , o f the t h r e e d i a b e t i c groups , hear t d y s f u n c t i o n was present o n l y in the S H D and D I A r a t s , w h e r e a s „ • W K D r a t s d i d not show any d e p r e s s i o n in c a r d i a c f u n c t i o n . S i m i l a r l y , o n l y i n the case of S H D and D I A r a t s w e r e r e d u c e d l e v e l s o f T ^ seen, w h e r e a s W K D r a t s d i d not show any change i n T ^ l e v e l s w h e n c o m p a r e d to t h e i r c o n t r o l s . T h e s i g n i f i c a n c e o f th is r e s u l t is d i f f i c u l t to assess as e a r l i e r 93 s tudies have shown that t r e a t m e n t of d i a b e t i c a n i m a l s w i t h p h y s i o l o g i c a l doses o f t h y r o i d h o r m o n e f a i l e d to r e v e r s e d i a b e t i c - i n d u c e d depress ions i n c a r d i a c f u n c t i o n , m y o s i n A T P a s e , and s a r c o p l a s m i c r e t i c u l a r C a 2 + pump a c t i v i t i e s ( G a n g u l y et a l . , 1983; G a r b e r et a l . , 1983; T a h i l i a n i and M c N e i l l , 1984). H o w e v e r , P i e r c e and D h a l l a (1985) have p o i n t e d out the p o s s i b i l i t y that a r e d u c t i o n in the n u m b e r of t h y r o i d h o r m o n e r e c e p t o r s m a y a t t e n u a t e the p h y s i o l o g i c a l e f f e c t s o f t h y r o i d h o r m o n e i n the d i a b e t i c a n i m a l . T h i s sugges t ion is s u b s t a n t i a t e d by the r e p o r t of D i l l m a n (1982), w h o s t u d i e d the e f f e c t of t h y r o i d r e p l a c e m e n t t h e r a p y on m y o s i n A T P a s e i n d i a b e t i c r a t s . W h i l e a p h y s i o l o g i c a l dose of T ^ (3ug.kg ^ .day *) was not e f f e c t i v e in n o r m a l i z i n g c a r d i a c m y o s i n A T P a s e , a l a r g e r p h a r m a c o -l o g i c a l dose (30 u g . k g * . d a y *) s u c c e s s f u l l y r e v e r s e d the d e p r e s s i o n to n o r m a l . O f f u r t h e r i n t e r e s t are r e c e n t e x p e r i m e n t s f r o m our l a b o r a t o r y us ing the c o m b i n e d t r e a t m e n t o f d i a b e t i c a n i m a l s w i t h t r i i o d o t h r y o n i n e and m e t h y l p a l m o x i r a t e ( T a h i l i a n i and M c N e i l l , 1985) and those o f P e n p a r g k u l et a l . (1980) us ing c o n t i n u o u s T ^ a d m i n i s t r a t i o n w h i c h d e m o n -s t r a t e at least p a r t i a l p r e v e n t i o n o f the adverse e f f e c t o f d i a b e t e s on hear t f u n c t i o n . Thus , i t appears t h a t the absence of m y o c a r d i a l d y s f u n c -t i o n i n W K D r a t s m a y be c o n s e q u e n c e of n o r m a l c i r c u l a t i n g t h y r o i d h o r m o n e in these d i a b e t i c a n i m a l s . These r e s u l t s , t o g e t h e r w i t h r e p o r t s tha t b o t h h y p o t h y r o i d i s m (Rodgers et a l . , 1982) and d i a b e t e s ( J a c k s o n and M c N e i l l , 1983b) r e s u l t i n an e n h a n c e d response to a l p h a - a d r e n e r g i c and a d e c r e a s e d response to b e t a - a d r e n e r g i c a m i n e s and a r e c e n t r e p o r t by A t k i n s et a l . (1985) w h o suggested t h a t a l t e r a t i o n s i n b e t a - a d r e n e r g i c r e c e p t o r s and t h e i r c o u p l i n g to a d e n y l a t e c y c l a s e m a y be i m p o r t a n t i n the d e v e l o p m e n t of d i a b e t i c c a r d i o m y o p a t h y , w o u l d i n d i c a t e that a c l o s e r 94 look s h o u l d be t a k e n at the poss ib le c o n n e c t i o n b e t w e e n d i a b e t e s - i n d u c e d c a r d i o m y o p a t h y and the i n f l u e n c e s o f a t t e n d a n t h y p o t h y r o i d i s m . In a d d i -t i o n , a l t e r a t i o n s in l i p i d m e t a b o l i s m c a n a l t e r h e a r t f u n c t i o n by m o d i f y i n g the s t r u c t u r e of c a r d i a c p l a s m a and s u b c e l l u l a r m e m b r a n e s . F o r e x a m p l e , long c h a i n a c y l c a r n i t i n e s , w h i c h are e l e v a t e d i n the m i c r o s o m a l s a r c o p l a s -m i c r e t i c u l a r p r e p a r a t i o n s d e r i v e d f r o m c h r o n i c a l l y d i a b e t i c r a t s , a re k n o w n to i n h i b i t m e m b r a n e t r a n s p o r t p r o t e i n s i n c l u d i n g S R c a l c i u m A T P a s e ( L o p a s c h u k et a l . , 1983). T h i s p o t e n c y r e s u l t s f r o m the s t r o n g l y c h a r g e d c a r n i t i n e m o i e t y o f the m o l e c u l e w h i c h m a k e s i t an e x c e l l e n t a m p h i p h i l e . T h u s , the l a c k o f c a r d i a c d y s f u n c t i o n i n W K Y d i a b e t i c r a t s c o u l d a lso be e x p l a i n e d by the f a c t tha t these a n i m a l s have a n o r m a l l i p i d m e t a b o -l i s m . T h e h igh m o r t a l i t y i n our h y p e r t e n s i v e - d i a b e t i c a n i m a l s was s i m i l a r to that n o t e d by F e i n et a l . (1984) i n r e n o v a s c u l a r h y p e r t e n s i v e d i a b e t i c r a t s and , as i n t h e i r s t u d y , our a n i m a l s d i e d w i t h o u t obv ious w a r n i n g signs s u c h as a s i g n i f i c a n t f a l l in body w e i g h t or b l o o d p r e s s u r e . S i m i l a r l y , as i n t h e i r s tudy , the m a g n i t u d e of the c o n t r a c t i l e a l t e r a t i o n s i n the S H D r a t s w a s g e n e r a l l y g r e a t e r t h a n those i n m u s c l e s f r o m the o t h e r t w o d i a b e t i c groups . W h e t h e r the o b s e r v e d d i f f e r e n c e s i n c a r d i a c f u n c t i o n b e t w e e n the groups is due to the d i a b e t i c s t a t e per se or c a n be a c c o u n t e d f o r by m e t a b o l i c d i s t u r b a n c e s ( S H D a n i m a l s h a d the lowes t body w e i g h t ) is a m a j o r c o n c e r n . H o w e v e r , F e i n et a l . (1980) u n d e r f e d c o n t r o l r a t s to e q u a l the d i a b e t i c r a t body w e i g h t and s h o w e d that w e i g h t loss was not a p r i m a r y cause f o r changes t h e y o b s e r v e d . T h e p r e c i s e e t i o l o g y of th is i n c r e a s e d m o r t a l i t y and m a r k e d c a r d i a c d e p r e s s i o n i n s p o n t a n e o u s l y h y p e r t e n s i v e - d i a b e t i c r a t s r e m a i n s to be e l u c i d a t e d . H o w -95 e v e r , p u b l i s h e d d a t a f r o m m o r p h o l o g i c a l s tudies have shown a g r e a t e r i n t e r s t i t i a l and r e p l a c e m e n t f i b r o s i s ( F a c t o r et a l . , 1983) and areas o f m i c r o v a s c u l a r t o r t u o s i t y , f o c a l c o n s t r i c t i o n s and m i c r o a n e u r y s m ( F a c t o r et a l . , 1984) in r e n o v a s c u l a r h y p e r t e n s i v e - d i a b e t i c a n i m a l s than in e i t h e r c o n d i t i o n a l o n e . S i n c e s i m i l a r m o r p h o l o g i c a l r e s u l t s h a v e been o b t a i n e d i n the s p o n t a n e o u s l y h y p e r t e n s i v e - d i a b e t i c r a t ( H a s h i m o t o , 1969; l a m s and W e x l e r , 1977) i t is poss ib le t h a t these s t r u c t u r a l a l t e r a t i o n s m i g h t e x p l a i n our f u n c t i o n a l r e s u l t s . T h e present s tudy a lso d e s c r i b e s the e f f e c t of h y d r a l a z i n e in S T Z d i a b e t i c r a t s . D a i l y t r e a t m e n t w i t h h y d r a l a z i n e i m p r o v e d c a r d i a c p e r f o r -m a n c e i n S T Z d i a b e t i c r a t s . M o r e o v e r , u n t r e a t e d d i a b e t i c r a t s d e v e l o p e d h y p e r t e n s i o n and b r a d y c a r d i a and upon t r e a t m e n t w i t h h y d r a l a z i n e these e f f e c t s w e r e p r e v e n t e d . F u r t h e r m o r e , l o n g - t e r m d i a b e t e s , i n d u c e d by S T Z , e l e v a t e d s e r u m l i p i d l e v e l s . O u r r e s u l t s s h o w n t h a t h y d r a l a z i n e t r e a t m e n t r e s t o r e s these l e v e l s to n o r m a l . T r e a t m e n t w i t h S T Z r e s u l t e d i n a d i a b e t i c s t a t e c h a r a c t e r i z e d by h y p e r g l y c e m i a and loss of body w e i g h t as e a r l y as 3 days a f t e r t r e a t m e n t . S e r u m g l u c o s e l e v e l s w e r e found to be c o n s i s t e n t l y h igh i n a l l the r a t s r e c e i v i n g the d i a b e t o g e n w i t h c o r r e s p o n d i n g h y p o i n s u l i n e m i a . Insul in is a lso k n o w n to m o d i f y the b a l a n c e b e t w e e n r a t e s o f p r o t e i n synthes i s and d e g r a d a t i o n in a n u m b e r of t issues i n c l u d i n g the h e a r t . T a b l e 7 shows a l o w e r v a l u e f o r the body and h e a r t w e i g h t s o f u n t r e a t e d d i a b e t i c r a t s as c o m p a r e d t o c o n t r o l s . S i m i l a r a l t e r a t i o n s i n hear t w e i g h t and t o t a l body w e i g h t w e r e a lso r e p o r t e d by o t h e r i n v e s t i g a t o r s ( F e i n et a l . , 1980; V a d l a m u d i et a l . , 1982). H o w e v e r , e v e n at s i m i l a r degrees of h y p e r -g l y c e m i a a loss in h e a r t and l e f t v e n t r i c u l a r w e i g h t s i n h y d r a l a z i n e - t r e a t e d 96 d i a b e t i c r a t s d i d not o c c u r . L e f t v e n t r i c l e to r i g h t v e n t r i c l e r a t i o s w e r e e l e v a t e d o n l y in the u n t r e a t e d d i a b e t i c r a t s . T h e r e l a t i v e l y e n l a r g e d v e n t r i c l e suggests the presence o f v e n t r i c u l a r h y p e r t r o p h y i n these a n i m a l s . F u r t h e r m o r e , . . . h y d r a l a z i n e p r e t r e a t m e n t o f the d i a b e t i c r a t s l o w e r e d b l o o d pressure but had no e f f e c t on the e l e v a t e d g l u c o s e l e v e l s . S i m i l a r l y , the m a i n t e n a n c e o f the hear t r a t e i n our h y d r a l a z i n e - t r e a t e d d i a b e t i c group c o u l d be a t t r i b u t e d to the n o r m a l t h y r o i d s ta tus i n these a n i m a l s . A s d e s c r i b e d p r e v i o u s l y , the t h y r o i d s ta tus of an a n i m a l is a lso k n o w n to a f f e c t the c o n t r a c t i l e c a p a b i l i t i e s o f the h e a r t . A s s h o w n i n th is s t u d y , h y d r a l a z i n e p r e t r e a t m e n t of d i a b e t i c r a t s m a i n t a i n e d the t h y r o i d h o r m o n e l e v e l s to near n o r m a l v a l u e s w i t h a subsequent i m p r o v e m e n t i n c a r d i a c f u n c t i o n . C o n v e r s i o n of T ^ to T ^ i n l i v e r s l i c e s f r o m d i a b e t i c r a t s is r e d u c e d (Ba lsam et a l . , 1978) but c l e a r a n c e of T ^ and T ^ by m e t a -b o l i s m and e x c r e t i o n is n o r m a l (Boado et a l . , 1978). T h e poss ib le cause o f the d i a b e t e s - i n d u c e d h y p o t h y r o i d i s m c o u l d be a d e f e c t i v e c o n v e r s i o n o f T ^ to Ty In a r e c e n t s t u d y f r o m our l a b o r a t o r y i t w a s r e p o r t e d t h a t w h i l e T ^ t r e a t m e n t c o r r e c t s the l o w e r e d T ^ B i n d e x o b s e r v e d i n d i a b e t e s , T ^ l e v e l s are s t i l l u n c h a n g e d or d e c r e a s e d a f t e r T ^ t r e a t m e n t ( G o y a l et a l . , 1985). It is sugges ted t h a t th i s c o u l d be due to a d e c r e a s e i n s e r u m T S H l e v e l s as r e p o r t e d e a r l i e r ( J o l i n and G o n z a l e z , 1978). H y d r a l a z i n e t r e a t m e n t i n c r e a s e d b o t h T ^ B as w e l l as T ^ l e v e l s in d i a b e t i c r a t s (Tab le 2). T h e m e c h a n i s m of th i s a c t i o n is not u n d e r s t o o d at present but p r e s u m a b l y c o u l d be through T S H r e l e a s e and a c o r r e c t i o n of the T ^ to T g c o n v e r s i o n d e f i c i t . H y d r a l a z i n e is k n o w n to c a u s e t a c h y c a r d i a in a n i m a l s and h u m a n 97 beings ( W a l k e r et a l . , 1951; B e i n et a l . , 1953; A b l a d , 1963). T h i s e f f e c t is m a i n l y a t t r i b u t e d to the r e f l e x s t i m u l a t i o n o f c a r d i a c s y m p a t h e t i c nerves s i n c e i t c a n be r e d u c e d or a b o l i s h e d by b e t a - a d r e n o c e p t o r b l o c k a d e (Brunner et a l . , 1967). In a d d i t i o n to the r e f l e x s t i m u l a t i o n of s y m p a t h e -t i c n e r v e s , h y d r a l a z i n e is a lso r e p o r t e d to p r o d u c e d i r e c t a c t i o n on the hear t ( K h a t r i et a l . , 1977). In the present s t u d y h y d r a l a z i n e was f o u n d to cause a s m a l l i n c r e a s e i n the r a t e o f r i g h t a t r i u m at l o w e r doses, w h i c h was not s i g n i f i c a n t . H o w e v e r , at h i g h e r doses i t p r o d u c e d a n e g a -t i v e c h r o n o t r o p i c e f f e c t . S i m i l a r l y i n l e f t a t r i a , h y d r a l a z i n e was f o u n d to be i n e f f e c t i v e , e x c e p t at h igh doses that p r o d u c e d a s l i g h t p o s i t i v e i n o t r o p i c e f f e c t ( F i g . 13). These e f f e c t s w e r e not a l t e r e d i n d i a b e t i c p r e p a r a t i o n s . T h u s the p r e v e n t i o n of d i a b e t e s - i n d u c e d b r a d y c a r d i a and c a r d i a c d y s f u n c t i o n by h y d r a l a z i n e c o u l d not be e x p l a i n e d on the basis o f i t s d i r e c t a c t i o n s on the h e a r t . T h e p r o n o u n c e d e f f e c t of h y d r a l a z i n e i n l o w e r i n g b l o o d l i p i d s i n the S T Z d i a b e t i c r a t was unusual but not t o t a l l y u n e x p e c t e d s i n c e t h e r e w a s a p r e v i o u s s t u d y by P e r r y and M i l l s (1962). T e n w o m e n w i t h h igh c h o l e s t e r o l va lues but n o r m a l b l o o d pressure and w h o w e r e c o m p l a i n i n g o f m i l d c h e s t p a i n , w e r e g i v e n p r o g r e s s i v e l y i n c r e a s i n g doses of h y d r a -l a z i n e . P a t i e n t s u n d e r w e n t t h r e e s e p a r a t e p e r i o d s o f h y d r a l a z i n e t r e a t -m e n t i n w h i c h the doses r a n g e d f r o m 100 to 200 m g / d a y , 300 to 400 m g / d a y , and 500 to 800 m g / d a y . N o change was seen w h i l e on l o w e r doses o f h y d r a l a z i n e . H o w e v e r , on the m o d e r a t e and large dose t r e a t -m e n t s , the m e a n l e v e l s of t o t a l c h o l e s t e r o l f e l l . T h e d a t a f o r s e r u m c h o l e s t e r o l and t r i g l y c e r i d e i n the u n t r e a t e d d i a b e t i c r a t a g a i n d e m o n -s t r a t e the r e l a t i o n s h i p b e t w e e n e l e v a t e d l e v e l s of these l i p i d s and l o w 98 s e r u m i n s u l i n l e v e l s seen in these a n i m a l s . H o w e v e r , h y d r a l a z i n e t r e a t e d d i a b e t i c r a t s , a l t h o u g h t h e y s t i l l had l o w s e r u m i n s u l i n , d i d not have the e l e v a t e d s e r u m c h o l e s t e r o l and t r i g l y c e r i d e s seen i n the u n t r e a t e d d i a b e t i c g r o u p . Thus the a c t i o n of h y d r a l a z i n e on s e r u m l i p i d s of d i a b e t i c r a t s appears to be independent o f i n s u l i n . O u r r e s u l t s r e g a r d i n g the e f f e c t s of S T Z d iabe tes on s e r u m f r e e f a t t y ac ids i n u n t r e a t e d d i a b e t i c r a t s are c o n s i s t e n t w i t h those r e p o r t e d e a r l i e r i n our S H R s t u d y . H o w -e v e r , h y d r a l a z i n e - t r e a t e d d i a b e t i c r a t s f a i l e d to show the e l e v a t i o n o f f r e e f a t t y a c i d s seen in the u n t r e a t e d d i a b e t i c g r o u p . T h i s c o u l d a g a i n p a r t i a l l y e x p l a i n the l a c k of e l e v a t i o n i n s e r u m c h o l e s t e r o l and t r i g l y -c e r i d e that is o b s e r v e d in these r a t s . W h y s e r u m l i p i d l e v e l s are not e l e v a t e d in these r a t s e v e n in a s e t t i n g of i n s u l i n l a c k is u n k n o w n at th is s tage but a m e c h a n i s m of a c t i o n of h y d r a l a z i n e c a n be s p e c u l a t e d u p o n . L i p o l y t i c h o r m o n e s a c t i v a t e a d e n y l a t e c y c l a s e to s t i m u l a t e c A M P a c c u m u l a t i o n i n the a d i p o c y t e and a lso enhance C a 2 + u p t a k e i n the c e l l s . In c o n t r a s t , a n t i l i p o l y t i c h o r m o n e i n s u l i n decreases the c A M P a c c u m u l a t i o n and C a 2 + u p t a k e . Thus b o t h c A M P a c c u m u l a t i o n and c a l c i u m are r e g a r d e d as the m a j o r c o m p o n e n t s o f an i n t r a c e l l u l a r s i g n a l -l i n g s y s t e m that responds to e x t e r n a l s t i m u l i . R e c e n t e v i d e n c e has 2+ s h o w n t h a t the messenger e f f e c t s o f C a u s u a l l y a c t v i a i t s m o d u l a t i o n e f f e c t on the a c t i v i t y of s p e c i f i c r e g u l a t o r y e n z y m e s such as a d e n y l a t e c y c l a s e . T h e r e g u l a t o r y r o l e o f C a 2 + on the e n z y m e s y s t e m is now k n o w n to depend upon b i n d i n g to c a l m o d u l i n ( C a M ) and the C a 2 + - C a M c o m p l e x serves as the r e g u l a t o r y l i g a n d by e x e r t i n g s t i m u l a t o r y e f f e c t s o n the a c t i v a t i o n o f c A M P - d e p e n d e n t p r o t e i n k inases i n c e l l e x t r a c t s 99 c a p a b l e of p h o s p h o r y l a t i n g s u b s t r a t e p r o t e i n s i n c l u d i n g h o r m o n e - s e n s i t i v e l ipase ( K a w a i , 1985). T h e p h o s p h o r y l a t i o n and a c t i v a t i o n o f t r i g l y c e r i d e l ipase then r e s u l t s in the d e g r a d a t i o n o f the s t o r e d t r i g l y c e r i d e to f a t t y a c i d and g l y c e r o l ( F i g . 16). A l t h o u g h the m e c h a n i s m of a c t i o n of h y d r a -l a z i n e is not c l e a r l y u n d e r s t o o d , C h i b a et a l . (1983) r e p o r t e d that h y d r a -l a z i n e p r o d u c e s v a s o d i l a t i o n t h r o u g h a d i r e c t e f f e c t on v a s c u l a r s m o o t h m u s c l e w h i c h m a y r e s u l t at least i n p a r t f r o m i t s i n h i b i t o r y e f f e c t on v a s c u l a r C a ^ + f l u x e s . T h i s e f f e c t o f h y d r a l a z i n e on C a ^ + f l u x e s c o u l d then be one e x p l a n a t i o n f o r f a i l u r e i n these a n i m a l s to show e l e v a t e d s e r u m f r e e f a t t y a c i d s . It c o u l d a lso be s p e c u l a t e d that h y d r a l a z i n e m a y i n h i b i t l i p o l y s i s by d e c r e a s i n g i n t r a c e l l u l a r c y c l i c A M P . H o w e v e r , C h i b a e t a l . (1983) f a i l e d to o b s e r v e any change i n c y c l i c A M P and c y c l i c G M P l e v e l s at a s ing le o r a l dose o f h y d r a l a z i n e w h i c h l o w e r e d s y s t o l i c b l o o d pressure o f S H R by 50 m m H g . A n o t h e r e x p l a n a t i o n f o r the f a i l u r e o f the h y d r a l a z i n e - t r e a t e d d i a b e -t i c r a t s to show e l e v a t e d s e r u m l i p i d s c o u l d be due to i t s d i r e c t e f f e c t on c a t e c h o l a m i n e s a f f e c t i n g b o t h t h e i r synthes i s and r e l e a s e . H y d r a l a z i n e has been shown to i n h i b i t d o p a - d e c a r b o x y l a s e (Sano et a l . , 1966) and d o p a m i n e - b e t a - h y d r o x y l a s e in an e x t r a c t p r e p a r e d f r o m b o v i n e a d r e n a l g lands ( L i u et a l . , 1974). In a d d i t i o n , h y d r a l a z i n e p r o d u c e d a c o n c e n -3 t r a t i o n - d e p e n d e n t i n h i b i t i o n of the c o n v e r s i o n of ( H ) - d o p a m i n e to 3 ( H ) - n o r a d r e n a l i n e i n r a t i s o l a t e d a t r i a . S i m i l a r l y i n r a t s t r e a t e d w i t h 3 h y d r a l a z i n e , t h e r e was an i n h i b i t i o n o f the c o n v e r s i o n of ( H ) - d o p a m i n e 3 to ( H ) - n o r a d r e n a l i n e in the i n t a c t a t r i a in v i v o ( S o n g i t t i g u n a et a l . , 1980). F i n a l l y , C h e v i l l a r d et a l . (1980) have s h o w n that h y d r a l a z i n e s u p e r f u s i o n of i s o l a t e d p r o x i m a l s e g m e n t s of r a t t a i l a r t e r y i n h i b i t e d 100 v a s o c o n s t r i c t o r responses to t r a n s m u r a l s t i m u l a t i o n i n a c o n c e n t r a t i o n dependent m a n n e r and h y d r a l a z i n e is k n o w n to i n h i b i t the spontaneous r e l e a s e of n o r a d r e n a l i n e i n r a t h e a r t ( S o n g i t t i g u n a and R a n d , 1980). T h e f a l l in b l o o d pressure r e s u l t i n g f r o m d i r e c t v a s o d i l a t o r a c t i o n of h y d r a l a z i n e r e f l e x l y e l i c i t s i n c r e a s e d a c t i v i t y i n c a r d i a c and v a s o m o t o r s y m p a t h e t i c nerves to p r o d u c e t a c h y c a r d i a (Gross , 1977). Indeed, d u r i n g the f i r s t w e e k o f t r e a t m e n t w i t h h y d r a l a z i n e b o t h c o n t r o l and d i a b e t i c r a t s s h o w e d an i n c r e a s e in hear t r a t e . D u r i n g per iods of r a p i d n e r v e s t i m u l a t i o n , d o p a m i n e b e t a - h y d r o x y l a s e is the r a t e - l i m i t i n g e n z y m e i n the r e - s y n t h e s i s o f n o r a d r e n a l i n e . U n d e r these c o n d i t i o n s , an i n h i b i t o r of d o p a m i n e b e t a - h y d r o x y l a s e w o u l d h a v e a m o r e p r o f o u n d e f f e c t on n o r a d r e n a l i n e synthes i s t h a n i n the r e s t i n g n e r v e s . I n h i b i t i o n by h y d r a -l a z i n e o f the c o n v e r s i o n of d o p a m i n e to n o r a d r e n a l i n e m a y r e s u l t i n the a c c u m u l a t i o n and r e l e a s e o f d o p a m i n e w h i c h not o n l y is a w e a k e r agonis t but c a n have i n h i b i t o r y f e e d b a c k on subsequent t r a n s m i t t e r r e l e a s e (Hope et a l . , 1980).. T h e present r e p o r t of the e f f e c t s of h y d r a l a z i n e in S T Z d i a b e t i c r a t s appear to be i m p o r t a n t for the f o l l o w i n g reasons . N u m e r o u s s tudies h a v e i n d i c a t e d that some agents used to r e d u c e the r i s k s o f h ig h b l o o d p r e s s u r e m a y cause changes in m e t a b o l i c p a r a m e t e r s t h e m s e l v e s . In f a c t , f i r s t l i n e drugs c u r r e n t l y used f o r the t r e a t m e n t o f d i a b e t i c h y p e r -t e n s i o n are t h i a z i d e d i u r e t i c s w h i c h i n c r e a s e t r i g l y c e r i d e s and t o t a l c h o l e s t e r o l ( A m e s and H i l l , 1976) and p r o p r a n o l o l w h i c h c a u s e d e l e v a t i o n o f t r i g l y c e r i d e s ( T a n a k a et a l . , 1976). B o t h c l i n i c a l and p a t h o l o g i c s tudies i n d i c a t e tha t a t h e r o s c l e r o s i s deve lops f a s t e r and m o r e e x t e n s i v e l y i n the p r e s e n c e of d i a b e t e s ( R o b e r t s o n and S t r o n g , 1968) w i t h e l e v a t i o n s 101 of s e r u m l i p i d s as an i m p o r t a n t r i s k f a s t o r ( K a n n e l and M c G e e , 1979). O t h e r e x p e r i m e n t a l r e s u l t s a lso i n d i c a t e a poss ib le d e t e r i o r a t i n g i n f l u e n c e o f h i g h f r e e f a t t y a c i d c o n c e n t r a t i o n s on m y o c a r d i a l f u n c t i o n . O p i e (1970) r e p o r t e d that p e r f u s i o n of i s o l a t e d r a t h e a r t s w i t h a m e d i u m c o n t a i n i n g 2.8 m M - o c t a n o a t e p r o d u c e s A V c o n d u c t i o n d i s t u r b a n c e s . W i l l e b r a n d s (1973) p e r f u s e d i s o l a t e d r a t h e a r t s w i t h a b a l a n c e d sa l t s o l u -t i o n c o n t a i n i n g h u m a n a l b u m i n (0.66 m M ) and o l e i c a c i d and f o u n d that the a m p l i t u d e of c o n t r a c t i o n d e c r e a s e d m o r e r a p i d l y w i t h t i m e i n e x p e r i -m e n t s w i t h o l e i c a c i d t h a n i n those w i t h o u t . A s m e n t i o n e d p r e v i o u s l y , a l t e r a t i o n s i n l i p i d m e t a b o l i s m c a n a l t e r h e a r t f u n c t i o n by m o d i f y i n g the s t r u c t u r e of c a r d i a c p l a s m a and s u b c e l l u l a r m e m b r a n e s . F i n a l l y , s t u d i e s h a v e shown t h a t the c o m b i n a t i o n o f h y p e r t e n s i o n and d i a b e t e s m e l l i t u s p r o d u c e s g r e a t e r m y o c a r d i a l d y s f u n c t i o n t h a n w i t h e i t h e r disease a lone and is a s s o c i a t e d w i t h a s i g n i f i c a n t m o r t a l i t y ( F e i n et a l . , 1984; R o d r i g u e s et a l . , 1985). In our S H R s t u d y , w e o b s e r v e d that m a l e W i s t a r r a t s s h o w e d a c a r d i a c depress ion at a l l l e f t a t r i a l f i l l i n g pressures w h e n c o m p a r e d to t h e i r c o n t r o l s . T h i s was c o n t r a r y to p r e v i o u s r e s u l t s f r o m our l a b o r a -t o r y us ing f e m a l e W i s t a r r a t s w h e r e c a r d i a c depress ion was o b s e r v e d o n l y at h igh l e f t a t r i a l f i l l i n g pressures . T h i s s tudy d e s c r i b e s the e f f e c t of S T Z - i n d u c e d d iabe tes on c a r d i a c f u n c t i o n i n m a l e and f e m a l e r a t s . S e r u m g l u c o s e was found to be c o n s i s t e n t l y h igh in a l l the r a t s r e c e i v i n g the d i a b e t o g e n . H o w e v e r , e v e n a s i m i l a r degrees o f h y p e r g l y c e m i a , loss i n h e a r t w e i g h t in f e m a l e d i a b e t i c r a t s d i d not o c c u r . S i m i l a r l y , body g r o w t h was suppressed to a g r e a t e r e x t e n t in m a l e d i a b e t i c r a t s . D r y h e a r t w e i g h t to body w e i g h t r a t i o s w e r e e l e v a t e d o n l y i n the m a l e 102 d i a b e t i c r a t s and m i g h t i m p l y the presence o f c a r d i a c h y p e r t r o p h y i n these a n i m a l s . O n e o f the m o s t p r o m i n e n t f e a t u r e s o f h e a r t s f r o m m a l e d i a b e t i c r a t s was the i n a b i l i t y to a c h i e v e +dP/dt va lues e q u i v a l e n t to those i n h e a r t s f r o m m a l e c o n t r o l s at a l l l e f t a t r i a l f i l l i n g pressures or in hear t s f r o m f e m a l e d i a b e t i c s at f i l l i n g pressures g r e a t e r than 7.5 c m F ^ O . T h e o b s e r v a t i o n of s i g n i f i c a n t d i f f e r e n c e s i n m u s c l e f u n c t i o n suggests t h a t the c a r d i a c e f f e c t s of S T Z are g r e a t e r i n the m a l e r a t . T h e l o w e r +dP/dt v a l u e s i n m a l e d i a b e t i c r a t s c o m p a r e d to f e m a l e d i a b e t i c s was not due to d i f f e r e n c e s in body w e i g h t and hear t s i z e s i n c e m a l e d i a b e t i c s had the l a r g e r body w e i g h t s and hear t mass . N e i t h e r is the g r e a t e r loss i n body w e i g h t in m a l e d i a b e t i c s a m a j o r c o n c e r n . F e i n et a l . (1980) u n d e r f e d c o n t r o l r a t s to e q u a l the d i a b e t i c r a t body weight* and s h o w e d that w e i g h t loss was not a p r i m a r y cause f o r the changes i n c a r d i a c f u n c t i o n that t h e y o b s e r v e d . W h e t h e r the d i a b e t i c e f f e c t s on the m y o c a r d i u m are r e l a t e d to the b l o o d g luc ose or to the a m o u n t o f i n s u l i n is d e b a t a b l e . R u b i n s t e i n et a l . (1984) have r e p o r t e d that d i a b e t i c r a t s t r e a t e d w i t h i n s u l i n , but i n w h i c h b l o o d g luc ose was kept at h i g h l e v e l s , d e m o n s t r a t e d c o r r e c t i o n o f b o t h m e c h a n i c s in i s o l a t e d p a p i l l a r y m u s c l e s and a c t o m y o s i n m e a s u r e -m e n t d e s p i t e the p e r s i s t e n c e o f h y p e r g l y c e m i a . T h a t h y p o i n s u l i n e m i a and not h y p e r g l y c e m i a is the m o r e i m p o r t a n t f a c t o r in d i a b e t i c c a r d i o -m y o p a t h y is also sugges ted by the present r e s u l t s . W e o b s e r v e d h igher c o n c e n t r a t i o n s o f i n s u l i n i n n o n - d i a b e t i c m a l e s c o m p a r e d to n o n - d i a b e t i c f e m a l e r a t s , an a d v a n t a g e lost w i t h the d e v e l o p m e n t o f d i a b e t e s . It is poss ib le tha t t e s t o s t e r o n e induces e n z y m e s w h i c h a l t e r the S T Z m o l e -103 c u l e r e n d e r i n g i t m o r e b i o l o g i c a l l y a c t i v e and c y t o t o x i c to the Islets ( M a c L a r e n et a l . , 1980) or tha t e s t r o g e n a c c e l e r a t e s the h y d r o l y t i c de-g r a d a t i o n o f S T Z by a c t i v a t i o n or i n d u c t i o n of r e l e v a n t e n z y m e s (Pa ik et a l . , 1982). E s t r o g e n c o u l d a lso b l o c k the a c t i o n o f S T Z d i r e c t l y by i n t e r f e r i n g w i t h the t o x i n ' s i n t e r a c t i o n w i t h the t a r g e t c e l l s u r f a c e . T h e e n t r y o f S T Z i n t o b e t a c e l l s depends o n i t s g lucose m o i e t y , and g l ucose t r a n s p o r t o f some c e l l t y p e s is r a p i d l y and r e v e r s i b l y i n h i b i t e d by 1 7 - b e t a - e s t r a d i o l ( G a y and H e l f , 1980). In a d d i t i o n , e x p e r i -m e n t a l l y - i n d u c e d d i a b e t e s i n m a l e r a t s has been r e p o r t e d to be a s s o c i a t e d w i t h r e d u c e d s e r u m l e v e l s of g o n a d o t r o p i n s and t e s t o s t e r o n e ( J a c k s o n and H u t s o n , 1984) and sex h o r m o n e s are k n o w n to be i n v o l v e d i n r e g u l a -t i o n of c a r d i a c f u n c t i o n . In f a c t , S c h a i b l e et a l . (1984) have shown that the e f f e c t s of g o n a d e c t o m y on c a r d i a c f u n c t i o n and m y o s i n A T P a s e a c t i v i t y are o f g r e a t e r m a g n i t u d e i n m a l e t h a n in f e m a l e r a t s . W h e t h e r th is sex d i f f e r e n c e is due to a l t e r a t i o n i n t h y r o i d s ta tus or l i p i d m e t a b o -l i s m is not e v i d e n t f r o m our r e s u l t s . O t h e r d a t a i n d i c a t e tha t S T Z i t s e l f is not c a r d i o t o x i c ( F e i n et a l . , 1980). T h e present r e p o r t of s e v e r e f u n c t i o n a l d e r a n g e m e n t s and s i g n i f i c a n t m o r t a l i t y i n s p o n t a n e o u s l y h y p e r t e n s i v e - d i a b e t i c r a t s a l o n g w i t h s i m i l a r f i n d i n g s i n h u m a n s and r e n o v a s c u l a r h y p e r t e n s i v e - d i a b e t i c r a t s w o u l d suggest a d e q u a t e d e t e c t i o n and an e a r l y and e f f e c t i v e a n t i h y p e r t e n s i v e t r e a t m e n t o f h igh b l o o d pressure m a y be a m o n g the i m p o r t a n t f i e l d s o f p r e v e n t i v e c a r e i n d i a b e t e s m e l l i t u s . It w i l l o b v i o u s l y be of i n t e r e s t i n f u t u r e s tudies to e x p l o r e the m e c h a n i s m of a c t i o n of h y d r a l a z i n e on l i p i d m e t a b o l i s m but c e r t a i n l y , a l t e r a t i o n s in l i p i d l e v e l s p r o d u c e d by h y d r a l a z i n e m a y be b e n e f i c i a l and a d d i t i v e to the l o w e r i n g o f b l o o d 104 pressure i n the p r e v e n t i o n of c o r o n a r y and o t h e r a t h e r o s c l e r o t i c diseases seen d u r i n g d i a b e t e s . S i n c e i t is i m p o r t a n t to t r e a t d i a b e t i c h y p e r t e n s i o n w i t h drugs t h a t n e i t h e r cause nor w o r s e n h y p e r l i p i d e m i a , the a n t i h y p e r -t e n s i v e d r u g h y d r a l a z i n e c o u l d b e c o m e an i m p o r t a n t agent e s p e c i a l l y i f i t s l i p i d e f f e c t pers i s t s w h e n the d r u g is t i t r a t e d to a c h i e v e o p t i m a l a n t i h y p e r t e n s i v e response i n d i a b e t i c p a t i e n t s . 105 C O N C L U S I O N S 1. T h e c o m b i n a t i o n o f d i a b e t e s m e l l i t u s and s y s t o l i c h y p e r t e n s i o n p r o d u c e s p r o f o u n d c a r d i o v a s c u l a r changes i n the r a t a s s o c i a t e d w i t h an i n c r e a s e d m o r t a l i t y . 2. T h e degree of these changes v a r i e s w i t h the d u r a t i o n o f the h y p e r t e n -s i v e - d i a b e t i c s t a t e . 3. E x p e r i m e n t a l l y i n d u c e d d i a b e t e s d i d not a l t e r the c a r d i a c f u n c t i o n i n W K D r a t s , w h e r e a s the c a r d i a c f u n c t i o n o f the S H D was most s e v e r e l y a f f e c t e d . S i n c e the e s s e n t i a l l y u n a f f e c t e d W K D r a t s and the s e v e r e l y a f f e c t e d D I A and S H D r a t s r e c e i v e d the s a m e S T Z dose and a t t a i n e d the s a m e degree o f h y p e r g l y c e m i a , i t is not l i k e l y that these e f f e c t s w e r e due to a d i f f e r e n c e in s e v e r i t y o f the h y p e r g l y c e m i a i n the t h r e e groups . 4. A l t h o u g h W K D r a t s w e r e not h y p o t h y r o i d and d i d not h a v e e l e v a t e d s e r u m l i p i d l e v e l s , r e s u l t s w h i c h c o u l d p o s s i b l y e x p l a i n the l a c k o f c a r d i a c d e p r e s s i o n , the n a t u r e and p h y s i o l o g i c a l s i g n i f i c a n c e o f these o b s e r v a t i o n s have y e t to be d e t e r m i n e d . 5. S i m i l a r l y , h y d r a l a z i n e t r e a t m e n t o f e x p e r i m e n t a l l y i n d u c e d d i a b e t i c r a t s i m p r o v e d c a r d i a c f u n c t i o n . W h e t h e r th is i m p r o v e m e n t was due to the pressure or l i p i d l o w e r i n g e f f e c t o f h y d r a l a z i n e or to i t s m a i n t e -nance o f t h y r o i d s ta tus has y e t to be e x p l o r e d . 6. 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H . : E f f e c t s of h y d r a l a z i n e on s t r e p t o z o t o c i n - i n d u c e d d i a b e t i c r a t s . 1. P r e v e n t i o n o f h y p e r l i p i d e m i a and i m p r o v e m e n t i n c a r d i a c f u n c t i o n ( s u b m i t t e d f o r p u b l i c a t i o n ) . H e y l i g e r , C . E . , R o d r i g u e s , B . and M c N e i l l , J . H . : E f f e c t o f c h o l i n e and m e t h i o n i n e t r e a t m e n t on c a r d i a c d y s f u n c t i o n o f d i a b e t i c r a t s ( s u b m i t t e d f o r p u b l i c a t i o n ) . G o y a l , R . K . , R o d r i g u e s , B . and M c N e i l l , J . H . : E f f e c t s of h y d r a l a z i n e on s t r e p t o z o t o c i n - i n d u c e d d i a b e t i c r a t s . 2. A l t e r a t i o n in c a r d i a c respons iveness to a d r e n e r g i c agonis ts ( s u b m i t t e d f o r p u b l i c a t i o n ) . G o y a l , R . K . , R o d r i g u e s , B . and M c N e i l l , J . H . : E f f e c t o f T 3 on the c a r d i a c responses to a d r e n e r g i c agonis ts i n S T Z - i n d u c e d d i a b e t i c r a t s ( s u b m i t t e d for p u b l i c a t i o n ) . 

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