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The effect of streptozotocin-induced diabetes on the male Wistar rat : hepatic high capacity, low affinity… Smith, D'Arcy Randall 1986

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c . I THE EFFECT OF STREPTOZOTOCIN-INDUCED DIABETES ON THE MALE WISTAR RAT HEPATIC HIGH C A P A C I T Y , LOW A F F I N I T Y ESTROGEN BINDING PROTEIN by D ' A r c y R a n d a l l S m i t h B . S c , U n i v e r s i t y o f A l b e r t a , 1982 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES FACULTY OF PHARMACEUTICAL SCIENCES UNIVERSITY OF BR IT ISH COLUMBIA We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BR I T ISH COLUMBIA F e b r u a r y , 1986 © D ' A r c y R a n d a l l S m i t h , 1986 In p resen t ing this thesis in partial fu l f i lment of t h e requ i rements for an a d v a n c e d d e g r e e at t h e Univers i ty of Brit ish C o l u m b i a , I agree that the Library shall m a k e it freely avai lable for re fe rence a n d s tudy . I further agree that p e r m i s s i o n for ex tens ive c o p y i n g o f this thesis for scho la r l y p u r p o s e s may be g ran ted by the h e a d of m y d e p a r t m e n t o r by his o r her representat ives . It is u n d e r s t o o d that c o p y i n g o r p u b l i c a t i o n o f this thesis fo r f inancia l gain shall not b e a l l o w e d w i t h o u t m y wr i t ten p e r m i s s i o n . D e p a r t m e n t T h e Un ivers i ty o f Brit ish C o l u m b i a 1956 M a i n M a l l V a n c o u v e r , C a n a d a V 6 T 1Y3 DE-6 (3 /81) 1 1 ABSTRACT Severa l p a r a l l e l s have been noted between a r y l hydrocarbon hydroxy lase (AHH) a c t i v i t y (a measure of drug and s t e r o i d metabol ism) and the high c a p a c i t y low a f f i n i t y (HCLA) es t rogen b i n d i n g p r o t e i n . These i n c l u d e a sex and age dependency, as wel l as p a r a l l e l changes in the AHH and HCLA l e v e l s due to v a r i o u s p h y s i o l o g i c a l m a n i p u l a t i o n s ( e . g . gonadectomy and hypophysec tomy) . It has been suggested from t h i s ev idence that there i s a r e g u l a t o r y a c t i o n of the HCLA es t rogen b i n d i n g p r o t e i n on h e p a t i c AHH a c t i v i t y . S ince s t r e p t o z o t o c i n (STZ) induced d i a b e t e s i s known to cause a l t e r a t i o n s in AHH a c t i v i t y we i n v e s t i g a t e d the e f f e c t s of t h i s c o n d i t i o n on the HCLA est rogen b i n d i n g p r o t e i n , and sought the hormonal c o n t r o l mechanism f o r the HCLA es t rogen b i n d i n g p r o t e i n in t h i s s t a t e . At both four and ten days p o s t - i n d u c t i o n of d i a b e t e s with STZ, there was approx imate ly a 50% decrease in the b i n d i n g c a p a c i t y of the HCLA es t rogen b i n d i n g p r o t e i n , with no a l t e r a t i o n s in the Kd v a l u e . Hormonal replacement was undertaken to r e s t o r e the normal p h y s i o l o g i c a l l e v e l s of t e s t o s t e r o n e , i n s u l i n , t r i i o d o t h y r o n i n e , and growth hormone; a l l of which are depressed in the d i a b e t i c . None of the t reatment regimens c a r r i e d out were ab le to r e s t o r e the reduced b i n d i n g c a p a c i t y of the HCLA b i n d i n g p r o t e i n . Treatment with t e s t o s t e r o n e and i n s u l i n had p r e v i o u s l y been shown to r e s t o r e AHH a c t i v i t y in gonadectomized and d i a b e t i c i i i r a t s , r e s p e c t i v e l y . S i n c e we were u n a b l e t o r e s t o r e HCLA b i n d i n g p r o t e i n l e v e l s w i t h t h e s e t r e a t m e n t s i n t h e d i a b e t i c r a t , we c o n c l u d e t h a t t h e r e i s no d i r e c t r e g u l a t o r y a c t i o n o f t h e HCLA b i n d i n g p r o t e i n on AHH a c t i v i t y i n t h e r a t . S e v e r a l o t h e r s p e c i e s we re a l s o e x a m i n e d f o r t h e p r e s e n c e o f a h e p a t i c HCLA e s t r o g e n b i n d i n g p r o t e i n . We we r e u n a b l e t o d e t e c t any s u ch c omponen t i n any o f t h e o t h e r s p e c i e s e x a m i n e d , i n d i c a t i n g t h a t t h e HCLA b i n d i n g p r o t e i n may be r a t s p e c i f i c . What p h y s i o l o g i c a l r o l e t h e HCLA b i n d i n g p r o t e i n may be p l a y i n g r e m a i n s u n c l e a r a t t h i s t i m e . G a i l D. B e l l w a r d , P h . D . S u p e r v i s o r i v TABLE OF CONTENTS PAGE ABSTRACT i i LIST OF TABLES v i i LIST OF FIGURES v i i i LIST OF ABBREVIATIONS ix ACKNOWLEDGEMENT xi INTRODUCTION 1 1. D iabetes m e l l i t u s 1 2. Hepat i c es t rogen b i n d i n g p r o t e i n s 6 a . Es t rogen r e c e p t o r 6 b. HCLA b i n d i n g p r o t e i n 8 3. Hypothes is 13 MATERIALS AND METHODS 15 1. Chemicals and reagents 15 2. Animals 15 3. Animal t reatments 16 V 4. High c a p a c i t y , low a f f i n i t y b i n d i n g p r o t e i n assay 18 5. De te rmina t ion of p r o t e i n content 20 6. S t a t i s t i c a l a n a l y s i s 21 RESULTS 22 1. E f f e c t of p o s s i b l e b lood contaminat ion 22 2. E f f e c t of 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 e s 22 3. E f f e c t of i n s u l i n replacement in the d i a b e t i c animal 29 4. E f f e c t of subcutaneous i n j e c t i o n s 32 5. E f f e c t of hormone replacement 32 a . T e s t o s t e r o n e enanthate 32 b. 3 , 3 , 5 - t r i i o d o t h y r o n i n e 34 c . Growth hormone 34 6. E f f e c t of i n t ravenous growth horomone a d m i n i s t r a t i o n 40 a . Vena cava c a n n u l a t i o n 40 b. T a i l ve in a d m i n i s t r a t i o n in the c o n s c i o u s animal 42 7. Other s p e c i e s 46 8. S t a b i l i t y s t u d i e s 46 v i DISCUSSION 50 1. B ind ing k i n e t i c s 5 0 2. E f f e c t of STZ- induced d i a b e t e s 50 3. Hormone replacement 53 a . I n s u l i n 53 b. T e s t o s t e r o n e 53 c . T r i i o d o t h y r o n i n e 54 d . Growth hormone 54 4. Other s p e c i e s 58 PROPOSED FUTURE EXPERIMENTS 60 SUMMARY 62 BIBLIOGRAPHY 63 APPENDIX I 70 1) S a t u r a t i o n curves 70 v i i LIST OF TABLES PAGE Table I The e f f e c t of l i v e r p e r f u s i o n volume on the HCLA b i n d i n g p r o t e i n . . . . 23 Table II The e f f e c t of STZ- induced d i a b e t e s and i n s u l i n replacement on the HCLA b i n d i n g p r o t e i n 26 Tab le III The e f f e c t of subcutaneous i n j e c t i o n s on the HCLA b i n d i n g p r o t e i n 33 Table IV The e f f e c t of hormone replacement of the HCLA b i n d i n g p r o t e i n 35 Tab le V The e f f e c t of vena cava c a n n u l a t i o n on the HCLA b i n d i n g p r o t e i n 41 Table VI The e f f e c t of hormone replacement vi a t a i l ve in i n j e c t i o n in the consc ious animal on the HCLA b i n d i n g p r o t e i n . . . . 43 Table VII Other s p e c i e s examined f o r the h e p a t i c HCLA b i n d i n g p r o t e i n 48 Tab le VIII S t a b i l i t y study of the HCLA b i n d i n g p r o t e i n 49 v i i i LIST 'OF FIGURES PAGE F igure 1 E f f e c t of l i v e r p e r f u s i o n volume on the HCLA b i n d i n g p r o t e i n 25 F i g u r e 2 E f f e c t of STZ- induced d i a b e t e s on the HCLA b i n d i n g p r o t e i n 28 F i g u r e 3 E f f e c t of i n s u l i n replacement on the HCLA b i n d i n g p r o t e i n in the d i a b e t i c animals 31 F i g u r e 4 E f f e c t of t e s t o s t e r o n e and 3 , 3 , 5 - t r i i o d o t h y r o n i n e replacement in the d i a b e t i c animals 37 F i g u r e 5 E f f e c t of growth hormone replacement on the HCLA b i n d i n g p r o t e i n 39 F i g u r e 6 E f f e c t of t a i l ve in i n j e c t i o n s in consc ious animals on the HCLA b i n d i n g p r o t e i n 45 F i g u r e A l S a t u r a t i o n curve of c o m p e t i t i v e 71 b i n d i n g a s s a y , c o n t r o l F i g u r e A2 S a t u r a t i o n curve of c o m p e t i t i v e b i n d i n g assay , 4 day STZ- induced d i a b e t i c 73 1 X LIST OF ABBREVIATIONS AHH BB Bmax BSA cpm DCC dpm DTT EDTA e s t r a d i ol F GH HCLA i . v . Kd NSB pmol/mg PZI SB s . c . STZ T 3 T . C . D . D . TED ary l hydrocarbon h y d r o x y l a s e b io breed ing maximum b i n d i n g c a p a c i t y bovine serum albumin counts per minute dextran coated charcoa l d i s i n t e g r a t i o n s per minute di t h i o t h r e i t o l e t h y l ened i amine t e t r a a c e t i c ac id A l , 3 , 5 ( 1 0 ) _ e s t r a t r i e n _ 3 ) 1 7 p _ d 1 o 1 f r e e s t e r o i d c o n c e n t r a t i o n growth hormone high c a p a c i t y , low a f f i n i t y i n t ravenous e q u i l i b r i u m d i s s o c i a t o n cons tant n o n s p e c i f i c b i n d i n g p icomoles per m i l l i g r a m protamine z i n c i n s u l i n s p e c i f i c b i n d i n g subcutaneous s t r e p t o z o t o c i n 3 , 3 , 5 - t r i i o d o t h y r o n i n e 2 , 3 , 7 , 8 - t e t r a c h o l o r o d i b e n z o - p - d i o x i n T r i s base , e thy lened iamine t e t r a a c e t i c a c i d , d i t h i o t h r e i t o l , sodium molybdate , g l y c r e r o l TES t e s t o s t e r o n e To . I t e s t o s t e r o n e 4 A - a n d r o s t e n - 1 7 8 - o l - 3 - o n e Toronto r e g u l a r i n s u l i n ACKNOWLEDGEMENT I would l i k e to express my s i n c e r e g r a t i t u d e to Dr . G a i l Be l lward f o r her guidance and p a t i e n c e throughout th course of t h i s r e s e a r c h . I would a l s o l i k e to thank my parents f o r t h e i r s u p p o r t i v e a t t i t u d e s and unders tand ing dur ing my academic and research t r a i n i n g . 1 INTRODUCTION 1) D iabetes m e l l i t u s D iabetes m e l l i t u s i s a d i s e a s e c h a r a c t e r i z e d by excess g lucose in the blood and u r i n e . I ts p reva lence in North America has been es t imated at 2% of the p o p u l a t i o n . C o m p l i c a t i o n s of d i a b e t e s i n c l u d e : a t h e r o s c l e r o s i s ; c o r o n a r y a r t e r y d i s e a s e , which i s the l e a d i n g cause of death amongst middle aged d i a b e t i c s ; p e r i p h e r a l v a s c u l a r d i s e a s e ; h y p e r t e n s i o n ; c e r e b r o v a s c u l a r d i s e a s e ; v i s u a l d i s o r d e r s , such as g laucoma, c a t a r a c t s , r e t i n o p a t h y , and b l i n d n e s s ; renal d i s e a s e ; p r o t e i n u r e a ; p e r i p h e r a l neuropathy; c o m p l i c a t i o n s dur ing pregnancy , such as e x c e s s i v e b i r t h w e i g h t s and t o x e m i a . Even though d i a b e t e s was i d e n t i f i e d as a d i s e a s e e n t i t y as e a r l y as 1500 B.C. in Egypt , i t i s on ly very r e c e n t l y that the e t i o l o g y has been r e a l i z e d to be m u l t i f a c t o r i a l . There appear to be s e v e r a l steps in the development of d i a b e t e s : a g e n e t i c p r e d i s p o s t i o n (probab ly two genes on chromosome 6 ) , in combinat ion with the i n f l u e n c e of environmenta l agents (such as c e l l t o x i n s or v i r u s e s ) which act as a m p l i f y i n g f a c t o r s , l e a d i n g to d i r e c t P - c e l l d e s t r u c t i o n vi a an autoimmune mechanism and/or the lack of p - c e l l r e g e n e r a t i o n a f t e r i n j u r y . Thus d i a b e t e s i s not a c t u a l l y one d i s e a s e but a group of syndromes. It r e f l e c t s a v a r i e t y of g e n e t i c and env i ronmenta l causes and a m p l i f y i n g f a c t o r s l e a d i n g to metabo l i c and t i s s u e changes 2 as c h r o n i c c o m p l i c a t i o n s s e c o n d a r y t o i n s u f f i c i e n t i n s u l i n a c t i v i t y ( F a j a n s e t a l . , 1 9 7 8 ) . The p r e v a l e n c e o f d i a b e t e s and t h e many m a n i s f e s t a t i o n s w h i c h i t c an t a k e i n t h e c l i n i c a l p i c t u r e make i t one o f t h e l e a d i n g h e a l t h c o n c e r n s t o d a y . R e s e a r c h i n t o d i a b e t e s has l e d t o many d i f f e r e n t a n i m a l m o d e l s o f d i a b e t e s b e i n g a v a i l a b l e ( f o r r e v i e w s see Mo r de s and R o s s i n i , 1 9 8 1 ; G r o d s k y e t a l . , 1 9 8 2 ; L i k e , 1985) bu t s i n c e no one model i s c a p a b l e o f p r e c i s e l y c o r r e s p o n d i n g t o any t y p e o f human d i a b e t e s t h e mode l s h o u l d be u sed t o e x a m i n e s p e c i f i c p h e n o t y p i c , p a t h o l o g i c , o r g e n o t y p i c e x p r e s s i o n s o f t h e d i s e a s e . T h e r e a r e a number o f a n i m a l m o d e l s a v a i l a b l e w h i c h s p o n t a n e o u s l y d e v e l o p d i a b e t e s . T h e s e i n c l u d e : C 5 7 B L / 6 J m i c e , Z u c k e r and BB r a t s , t h e C h i n e s e h a m s t e r , New Z e a l a n d w h i t e r a b b i t s , t h e K e e s h o u n d d o g , and t h e C e l e b e s b l a c k a p e . T h e s e m o d e l s a l l v a r y i n t h e s e v e r i t y and p a t h o g e n e s i s o f t h e d i a b e t e s t h e y d e v e l o p and n o t a l l a n i m a l s i n t h e c o l o n y w i l l become d i a b e t i c . Due t o d i f f i c u l t i e s w h i c h can a r i s e i n t r y i n g t o m a i n t a i n a c o l o n y o f s p o n t a n e o u s l y d i a b e t i c a n i m a l s ( o r i n t r y i n g t o o b t a i n them f r o m o t h e r s ) much r e s e a r c h has been done t o c r e a t e c h e m i c a l o r b i o l o g i c a l me thod s w h i c h w i l l c r e a t e a d i a b e t i c s t a t e i n r e s e a r c h a n i m a l s . The ma in me t hod s w h i c h have been d e v e l o p e d c e n t e r on t h e use o f c y t o t o x i c chemi c a l s . The f i r s t d i a b e t o g e n i c a g e n t u s e d f o r r e s e a r c h was a l l o x a n . I t was w i d e l y u s e d as t h e c l a s s i c a l d i a b e t o g e n 3 u n t i l s t r e p t o z o t o c i n (STZ) was i n t r o d u c e d i n 1959 (Vavra et  a l . , 1960) . S tud ies us ing STZ to induce d i a b e t e s , q u i c k l y showed tha t i t produced a lower m o r t a l i t y ra te than a l l o x a n ( H o f t i e z e r and C a r p e n t e r , 1973), i t had a lower general t o x i c i t y , and the c h a r a c t e r i s t i c l e s i o n s ( i . e . P - c e l l t o x i c i t y ) were more r e p r o d u c i b l e ( A r i s o n et al . , 1967 ; Junod et a l . , 1969). It was p o s s i b l e to produce a s e r i e s of d i a b e t i c s t a t e s of graded s e v e r i t y by v a r y i n g the dose (Junod et al . , 1969). Most i m p o r t a n t l y , the changes in t i s s u e m e t a b o l i c p a t t e r n s more c l o s e l y resemble those seen i n human d i a b e t e s m e l l i t u s (Mansford and O p i e , 1968) and the l e s i o n s produced by STZ most c l e a r l y resemble those seen in the i n s u l i t i s of human d i a b e t e s m e l l i t u s ( K l o p p e l , 1985). These f a c t o r s g ive weight to the v a l i d i t y of STZ- induced d i a b e t e s as a r e l e v a n t model f o r the d i s e a s e . Due to these f a c t o r s , a l l o x a n has l a r g e l y been r e p l a c e d by STZ as the main d i a b e t o g e n i c agent f o r e x p e r i m e n t a l l y induced d i a b e t e s m e l l i t u s , except in s t r a i n s h i g h l y r e s i s t a n t to STZ, such as the r a b b i t . The d i a b e t o g e n i c a c t i o n of STZ i s due to i t s s e l e c t i v e d e s t r u c t i o n of the p a n c r e a t i c 8 -ee l Is (Mordes et a l . , 1981). The d i a b e t i c s t a t e induced by STZ i s marked by 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 , h y p e r l i p e m i a , hyperke tonemia , and decreased growth and body weight (Montoya et a l . , 1974; Chen et a l . , 1982). There i s an i n v e r s e r e l a t i o n s h i p between s e n s i t i v i t y t o , and the s e v e r i t y o f , the d i a b e t o g e n i c p r o p e r t i e s of STZ and the age 4 of the animal ( M a s i e l l o et al . , 1979). There i s a l s o ev idence that the male animal i s more s e n s t i v e than the female in both s i n g l e dose (MacLeren et al . , 1980) and m u l t i p l e dose regimens ( R o s s i n i et al . , 1978). B e s i d e s p roduc ing a d i a b e t i c s t a t e , STZ a f f e c t s hormone s u p p l y and s e c r e t i o n . There i s a d e c r e a s e in i n s u l i n due to P - c e l l d e s t r u c t i o n . Serum t e s t o s t e r o n e i s s i g n i f i c a n t l y reduced in S T Z - t r e a t e d male animals (Baxter et a l . , 1981) to l e v e l s s i m i l a r to those seen in spontaneous ly d i a b e t i c male ra t s (Warren et a l . , 1983), wh i le S T Z - t r e a t e d female ra t s show an i n c r e a s e in serum t e s t o s t e r o n e to normal male l e v e l s (Learning et a 1 . , 1 9 8 2 ) . T h i s t e s t o s t e r o n e i n c r e a s e i s ma in ly of adrenal o r i g i n and c o r r e l a t e s with the s e v e r i t y of k e t o s i s . Thyro id d y s f u n c t i o n i s a l s o repor ted to be due to a decrease in the r e l e a s e of hypotha lamic t h y r o t r o p i n r e l e a s i n g hormone which causes p i t u i t a r y and t h y r o i d a l t e r a t i o n s . As w e l l , the plasma l e v e l s of t h y r o x i n , 3 ,3 , 5 - t r i i o d o t h y r o n i n e , and 3 , 3 - d i i o d o t h y r o n i n e f a l l , the plasma l e v e l of 3 , 5 - d i i o d o t h y r o n i n e i n c r e a s e s and there i s a decrease in the c o n v e r s i o n of t h y r o x i n to 3 ,3 , 5 - t r i i o d o t h y r o n i n e (Mi ts uma et a l . , 1982; J e n n i n g s , 1984; O r t i z - C a r o et a l . , 1 9 8 4 ) . A d e c r e a s e i n the r e l e a s e of growth hormone i s seen due to d e f i c i e n t s y n t h e s i s and s torage ( B l u e t - P a j o t et a l . , 1984). However, Tannenbaum (1981) measured normal l e v e l s of growth hormone in the p i t u i t a r y , thus p o i n t i n g to growth hormone r e l e a s e as the d e f i c i e n c y . The r e l e a s e p a t t e r n of growth hormone in the 5 male d i a b e t i c rat i s a l s o changed from i t s normal 'peak and t r o u g h ' ( u l t r a d i e n e ) r e l e a s e to the female ' c o n t i n u o u s l e v e l ' r e l e a s e p a t t e r n (Tannenbaum and M a r t i n , 1976; Tannenbaum, 1981). Th is a l t e r a t i o n in r e l e a s e p a t t e r n s seems to be due to i n c r e a s e d l e v e l s of s o m a t o s t a t i n in the S T Z - d i a b e t i c animal (Tannenbaum, 1981). Decreases in the c i r c u l a t i n g l e v e l s of l u i t e n i z i n g hormone and p r o l a c t i n are a l s o seen ( P e r e z - D i a z et a l . , 1982). STZ a l s o has d i r e c t e f f e c t s on the l i v e r . It produces d e g r a n u l a t i o n of the rough endoplasmic r e t i c u l u m and m i t o c h o n d r i a l s w e l l i n g wi th the l o s s of c r i s t e a , independent of whether or not d i a b e t e s i s produced (Languens et a l . , 1980). I ts a l s o been repor ted that o ther h e p a t i c changes i n c l u d e decreased w e i g h t , g lycogen c o n c e n t r a t i o n , c i t r i c ac id c o n t e n t , and i n c r e a s e d a c e t y l coA c o n t e n t , p r o t e i n s , and DNA p h o s p h o l i p i d P c o n c e n t r a t i o n (Montoya and H e r r e r a , 1974). Warren et a l . (1983) repor ted a l t e r a t i o n s in the p r o t e i n d i s t r i b u t i o n in the cytochrome P-450 r e g i o n . These a l t e r a t i o n s ( i n c r e a s e s in some bands and decreases in o t h e r s ) have a l s o been seen by other authors (Reinke et  a l . , 1978; Stohs et al . , 1979 ; Past and Cook, 1980; Peng et a l . , 1983) . O v e r a l l , the e f f e c t was to make drug metabol ism l e s s d i f f e r e n t i a t e d between the s e x e s . I n s u l i n t reatment was able to reverse the b iochemica l and metabo l i c a b n o r m a l i t i e s seen (Reinke et a l . , 1978; Reinke et a l . , 1979) . These changes in drug metabol ism were s i m i l a r in the STZ- induced d i a b e t i c animal and the spontaneous ly 6 d i a b e t i c animals (Warren et al . , 1983). 2) Hepat ic es t rogen b i n d i n g p r o t e i n s a) Es t rogen r e c e p t o r The f i r s t es t rogen b i n d i n g p r o t e i n to be found in the rat l i v e r was the es t rogen r e c e p t o r . Th is r e c e p t o r showed p h y s i c a l p r o p e r t i e s and b i n d i n g c h a r a c t e r i s t i c s which were i d e n t i c a l to the ' c l a s s i c a l ' es t rogen r e c e p t o r found in the u t e r u s : sed imenta t ion in the 8-9 S reg ion on sucrose d e n s i t y g r a d i e n t s ; s p e c i f i c i t y f o r s t e r o i d a l and n o n - s t e r o i d a l e s t r o g e n s ; high a f f i n i t y (10 M) and low c a p a c i t y (fmol/mg) f o r e s t r o g e n s ; and a s i m i l a r i s o e l e c t r i c f o c u s i n g p r o f i l e (Char iness et al . , 1975 ; V i l a d i u et a l . , 1975; E i s e n f e l d et al . , 1976; Beers and Rosner , 1977; Wrange et al . , 1 9 8 0 ) . The e s t r o g e n r e c e p t o r has a l s o been found in o ther t i s s u e s ( e . g . k i d n e y , hear t ) but at lower l e v e l s than in the l i v e r or uterus ( E i s e n f e l d et a l . , 1977; S i n g l e t a r y et a l . , 1 9 8 2 ) . The e s t r o g e n r e c e p t o r has a l s o been found in the l i v e r of both sexes of o ther s p e c i e s such as the r a b b i t , g reen monkey ( E i s e n f e l d et al . , 1 9 77 ) , and human (Duffy and D u f f y , 1978). The h e p a t i c es t rogen r e c e p t o r i s found in s i m i l a r l e v e l s in both male and female ra t s (Powel1-Jones et a l . , 1980; Thompson et a l . , 1981) and reaches i t s h i g h e s t l e v e l in the p o s t - p u b e r t a l animal (Rumbaugh et a l . , 1983). The h e p a t i c es t rogen r e c e p t o r in both sexes i s under 7 mu l t i ho rmona l r e g u l a t i o n . Gonadectomy causes an i n c r e a s e in the l e v e l s of r e c e p t o r in both male and female ra ts (Beers and Rosner , 1977 ; Eagon et al . , 1980; Powel l - Jones et  a l . , 1981), wh i le hypophysectomy causes a decrease in r e c e p t o r l e v e l s in both sexes (Norstedt et a l . , 1981 a; E r i k s s o n , 1982), as does adrenalectomy (Nors tedt et al . , 1981 a ) . The ev idence f o r the r e g u l a t o r y mechanism of the h e p a t i c es t rogen r e c e p t o r p o i n t s towards g l u c o c o r t i c o i d s in combinat ion with growth hormone and p r o l a c t i n as the p r o b a b l e a g e n t s (Chamness et al . , 19 75 ; N o r s t e d t et a 1. , 1981 a; Gusta fsson et a l . , 1983 ) . The h e p a t i c es t rogen r e c e p t o r in the c y t o s o l has been demonstrated to undergo nuc lear t r a n s l o c a t i o n where i t complexes with chromat in a f t e r i t has complexed with an e s t r o g e n i c l i g a n d . Th is occurs in both males and f e m a l e s , but i n males the t r a n s l o c a t i o n process i s s lower and n u c l e a r r e t e n t i o n longer ( E i s e n f e l d et a l . , 1976 ; Aten et a l . , 1978; Aten et a l . , 1980; Dickson and E i s e n f e l d , 1980). The l e v e l s of es t rogen r e c e p t o r have been c o r r e l a t e d with i n c r e a s e s in ren in s u b s t r a t e ( E i s e n f e l d et a l . , 19 7 6 ) , p lasma l e v e l s of very low d e n s i t y l i p o p r o t e i n s (Thompson et  a l . , 1983) , and an i n c r e a s e in the l e v e l s of the s p e c i f i c mRNA sequence f o r apo very low d e n s i t y 1 i p o p r o t e i n - 1 1 in av ian l i v e r (Snow et a l . , 1978). Recent ly debate has a r i s e n as to whether the es t rogen r e c e p t o r i s a c t u a l l y p r e s e n t in the c y t o s o l or i f i t t r u l y r e s i d e s in the nuc leus or on the nuc lear membrane ( i . e . i s the accepted model of 8 r e c e p t o r - 1 i g a n d complex ing in the c y t o s o l then t r a n s l o c a t i n g to the nuc leus to bind with chromat in based on an a r t i f a c t of exper imenta l procedure) (Gorsk i et a l • , 1984; King and Greene, 1984; S c h r a d e r , 1984). Th is i s an important q u e s t i o n to answer, but e i t h e r way i t w i l l not change the end r e s u l t that es t rogens do e l i c i t a s e r i e s of responses from the l i v e r which are mediated by es t rogen r e c e p t o r s . Not a l l of the h e p a t i c responses seen due to es t rogens can be accounted f o r by es t rogen r e c e p t o r - 1 i g a n d complexes i n t e r a c t i n g w i t h c h r o m a t i n ( T a m u l e v i c i u s et al . , 19 82 ; Lax et a l . , 1 9 8 3 ) . I t has been found t h a t e s t r o g e n s a l s o b ind with r e c e p t o r s that are a s s o c i a t e d with the plasma membrane ( P i e t r a s and Szego , 1979; 1980) , lysosomal membranes ( H i r s c h and Szego, 1974) , and microsomal membranes ( B l y t h et a l . , 1971; He l ton et a l . , 1977 ; Yamada and M i y a j i , 1982). B i n d i n g to these membranes cou ld cause e f f e c t s by a l t e r i n g 2 + i n t r a c e l l u l a r Ca c o n c e n t r a t i o n s and d i s t r i b u t i o n , or r e a r r a n g i n g some b i o l o g i c a l l y important components of the membrane or microsomal system. b) High c a p a c i t y , low a f f i n i t y b i n d i n g p r o t e i n Bes ides the es t rogen r e c e p t o r there i s a second c l a s s of e s t r o g e n b i n d i n g p r o t e i n in male rat l i v e r which s h a l l be r e f e r r e d to as the high c a p a c i t y , low a f f i n i t y (HCLA) b i n d i n g p r o t e i n . The HCLA b i n d i n g p r o t e i n has been repor ted by s e v e r a l groups to have b i n d i n g c h a r a c t e r i s t i c s which show lower 9 a f f i n i t y and h igher c a p a c i t y than those seen with r e c e p t o r sys tems . Values which have been repor ted a r e : Kd=10" 7 -- 8 -12 10" M , b i n d i n g c a p a c i t y 10" mol/mg p r o t e i n ( E a g o n et a l . , 1980); Kd = 1 0 " 7 - 1 0 " 8 M , b i n d i n g c a p a c i t y 1 0 " 1 3 mol/mg p r o t e i n (Rogerson and Eagon, 1984); Kd=10" 7 M, -15 b i n d i n g c a p a c i t y 10 mol/mg p r o t e i n (D ickson et a l . , - 8 - 1 1 1978); Kd=10" M, b i n d i n g c a p a c i t y 10" mol/mg p r o t e i n (Smirnov et a l . , 1977); Kd = 1 0 " 7 M , b i n d i n g c a p a c i t y -1 ? 10 mol/mg p r o t e i n (Warren, 1982; F i n l a y s o n , 1983). D i f f e r e n c e s in the va lues repor ted may be due to the v a r i o u s methodo log ies used to measure the p r o t e i n ( e . g . sucrose d e n s i t y g r a d i e n t s , gel f i l t r a t i o n , s i n g l e po int a n a l y s i s , Scatchard a n a l y s i s of m u l t i - p o i n t c o m p e t i t i v e b i n d i n g c u r v e s ) , d i f f e r e n c e s in the b u f f e r s used to prepare the c y t o s o l , and in the a n a l y s i s of the da ta o b t a i n e d . The HCLA b i n d i n g p r o t e i n sediments in the 3-4 S reg ion of sucrose d e n s i t y g r a d i e n t s (Smirnov et a l . , 1977 ; Eagon et a l . , 1980; Powel l - Jones et a l . , 1980) as compared to the e s t r o g e n r e c e p t o r which sediments in the 8-9 S reg ion (Eagon et a l . , 1980). The HCLA b i n d i n g p r o t e i n a l s o shows up as a unique peak in gel f i l t r a t i o n which i s not seen in the a n a l y s i s of c y t o s o l from adu l t female or prepubescent male and female ra ts (Dickson et al . , 1978; Eagon et al . , 1980 ; Thompson et al . , 1 9 8 1 ) . The HCLA b i n d i n g p r o t e i n i s capab le of b i n d i n g es t rogens and some androgens with the a f f i n i t y be ing h ighes t f o r s t e r o i d a l es t rogens ( e s t r i o l , e s t r a d i o l , e s t r o n e ) and approx imate ly 10-20 t imes l e s s f o r 1 0 the androgens which bind (2cc -hydroxy tes tos te rone , 5 a - a n d r o s t a n e - 3 a , 1 7 p - d i o 1 , 5 a - d i h y d r o t e s t o s t e r o n e ) (Miroshnechenko et a l . , 1983; Rogerson and Eagon, 1984). The important f u n c t i o n a l groups f o r b i n d i n g ( f o r both es t rogens and androgens) are the 3-a and 17-P hydroxy g roups , removal of which s h a r p l y decreases the b i n d i n g a f f i n i t y of the s t e r o i d (Miroshnechenko et a l . , 1983). The l e v e l s of the HCLA b i n d i n g p r o t e i n are much h igher in the mature male than in the mature f e m a l e . Thompson et  a l . (1981) repor ted a t e n - f o l d d i f f e r e n c e in l e v e l s , whi le Dickson et al . ( 1978) repor ted a two h u n d r e d - f o l d d i f f e r e n c e . Other groups have been unable to de tec t the HCLA b i n d i n g p r o t e i n in the mature female l i v e r (Eagon et  a l . , 1980; F i n l a y s o n , 1983). Thompson et a l . (1981) repor ted that sex d i f f e r e n c e s were not seen in the immature rat and the l e v e l s found in t h e i r l i v e r s were s i m i l a r to those seen in the mature f ema le . Powel l - Jones et a l . (1980) found that the HCLA b i n d i n g p r o t e i n was not seen in the immature male , but became ev ident a f t e r puberty (days 34-42) due to the t e s t o s t e r o n e surge seen in the male at t h i s t i m e . Th is t e s t o s t e r o n e surge i s the cue f o r the e x p r e s s i o n of the HCLA b i n d i n g p r o t e i n which has been n e o n a t a l l y i m p r i n t e d d u r i n g days 7-12. Th i s i s demonst ra ted by neonatal c a s t r a t i o n p r e v e n t i n g the development of the HCLA b i n d i n g p r o t e i n in the mature ma le , and t e s t o s t e r o n e t reatment of the neonatal c a s t r a t e s a l l o w i n g e x p r e s s i o n of normal male l e v e l s when these animals reach adu l thood (S loop 1 1 et a l . , 1983 ; Thompson and L u c i e r , 1983 ) . Once e x p r e s s e d , androgens are neccesary f o r the maintanence of the HCLA b i n d i n g p r o t e i n l e v e l s as demonstrated by c a s t r a t i o n of the adu l t male r e s u l t i n g in a t e s t o s t e r o n e - r e v e r s i b l e decrease in the HCLA b i n d i n g p r o t e i n l e v e l s ( F i n l a y s o n , 1983). In a d d i t i o n to the androgen dependency f o r e x p r e s s i o n and maintanence , the HCLA b i n d i n g p r o t e i n a l s o r e q u i r e s an i n t a c t p i t u i t a r y . Hypophysectomy of adu l t male and female r a t s r e s u l t e d in the a b o l i t i o n of sex d i f f e r e n c e s in the e x p r e s s i o n of the HCLA b i n d i n g p r o t e i n ( i . e . HCLA b i n d i n g p r o t e i n l e v e l s were decreased in the male and i n c r e a s e d in the female) (Powel1-Jones et a l . , 1980; L u c i e r et a l . , 1981; F i n l a y s o n , 1983). Growth hormone seems to be the agent p l a y i n g a r o l e in the sex d i f f e r e n c e s seen i n the HCLA b i n d i n g p r o t e i n l e v e l s . Whi le L u c i e r et a l . (1981) repor ted that growth hormone a d m i n i s t r a t i o n to hypophysectomized animals had no e f f e c t on HCLA b i n d i n g p r o t e i n l e v e l s , F i n l a y s o n (1983) repor ted a f u r t h e r 50-80% d e c r e a s e i n b i n d i n g c a p a c i t y i n the hypophysectomized animal (male and f e m a l e ) . Th i s r e p r e s s i o n of HCLA b i n d i n g p r o t e i n l e v e l s was a l s o seen by Rumbaugh et a l . (1983) when an e c t o p i c p i t u i t a r y was implanted in the hypophysectomized animal (male or f e m a l e ) . The main hormone(s) s e c r e t e d by an e c t o p i c p i t u i t a r y , which seemed to be r e s p o n s i b l e f o r the f e m i n i z a t i o n of HCLA b i n d i n g p r o t e i n l e v e l s , has not been i d e n t i f i e d with c e r t a i n t y but the ev idence p o i n t s to growth hormone. Growth hormone and p r o l a c t i n are s e c r e t e d in the 1 2 l a r g e s t amounts (Lam et al . , 1976 ; Gus ta fsson et al . , 1980) and s i n c e p r o l a c t i n had no measurable f e m i n i z i n g e f f e c t on o ther h e p a t i c parameters ( e . g . s t e r i o d metabol ism) (Eneroth et a l . , 1977) i t was conc luded there must be another " f e m i n i z i n g " f a c t o r being r e l e a s e d which was termed f e m i n o t r o p i n . Feminot rop in was l a t e r i d e n t i f i e d as growth hormone (Rumbaugh and C o l b y , 1980; Mode et a l . , 1983). The f e m i n i z i n g e f f e c t on HCLA b i n d i n g p r o t e i n l e v e l s of growth hormone s e c r e t e d from the e c t o p i c p i t u i t a r y is the same as that seen upon the a d m i n i s t r a t i o n of exogenous growth hormone ( F i n l a y s o n , 1983), which s t rengthens the p r o b a b i l i t y tha t growth hormone i s r e s p o n s i b l e f o r the decreased l e v e l . That growth hormone can have a f e m i n i z i n g e f f e c t on a s t e r o i d b i n d i n g system i s not a novel c o n c e p t . I ts a d m i n i s t r a t i o n to hypophysectomized male ra ts leads to female ( i . e . i n c r e a s e d ) c o n c e n t r a t i o n s of p r o l a c t i n r e c e p t o r s in the l i v e r (Nors tedt et a l . , 1981 b; Nors tedt et a l . , 1984). F u n c t i o n s f o r the HCLA b i n d i n g p r o t e i n have been suggested to be: the uptake and c o n c e n t r a t i o n of es t rogens and androgens (and t h e i r m e t a b o l i t e s ) thereby r e g u l a t i n g the d i s t r i b u t i o n between r e c e p t o r s and the enzymes of metabol ism (Miroshnechenko et a l . , 1982) , and the b i n d i n g and d i s p o s i t i o n of sex s t e r o i d s and t h e i r m e t a b o l i t e s (D ickson et a l . , 1978; Si n g l e t a r y et a l . , 1983). There i s a l s o the p o s s i b i l i t y that the HCLA b i n d i n g p r o t e i n may serve as a m o d i f y i n g mechanism f o r the t r a n s l o c a t i o n of the es t rogen 1 3 r e c e p t o r (Powel1-Jones et a l . , 1980) a l though i t does not show t r a n s l o c a t i o n i t s e l f (D ickson et a l . , 1978). An exper imenta l use f o r the HCLA b ind ing p r o t e i n may be as an i n d i c a t o r f o r the i n i t i a l s c r e e n i n g of male r e p r o d u c t i v e t o x i c a n t s , as demonstrated by the decreased l e v e l s ( f e m i n i z a t i o n ) in adu l t males (and m a s c u l i n i z a t i o n in adu l t females) exposed n e o n a t a l l y to t o x i c a n t s such as methyl mercury , 1 ,2 -d ib romo-3-ch io ropropane , and ch lo rdecone (Lawrence et a l . , 1984; L u i , 1985 ; Lawrence-Domey and L u i , 1985). 3) Hypothes is P rev ious work in our lab (Warren, 1982; F i n l a y s o n , 1983) has shown a number of c o r r e l a t i o n s between the HCLA es t rogen b i n d i n g p r o t e i n and the l e v e l s of a ry l hydrocarbon hydroxy lase (AHH) a c t i v i t y in the male r a t , r e f l e c t i n g the observed sexual d i f f e r e n c e s in drug and s t e r o i d metabo l i sm. These i n c l u d e : an age dependent sex d i f f e r e n c e in the l e v e l s of the HCLA b i n d i n g p r o t e i n and AHH a c t i v i t y ; gonadectomy produces a t e s t o s t e r o n e - r e v e r s i b l e decrease in the c a p a c i t y of the HCLA b i n d i n g p r o t e i n and in AHH a c t i v i t y ; hypophysectomy a b o l i s h e s the sex d i f f e r e n c e s seen in the c a p a c i t y of the HCLA b i n d i n g p r o t e i n and in AHH a c t i v i t y ; growth hormone a d m i n i s t r a t i o n to the hypophysectomized male or female rat causes a r e d u c t i o n in AHH a c t i v i t y and f u r t h e r decreases the c a p a c i t y of the HCLA b i n d i n g p r o t e i n . S ince t h e r e i s a r e d u c t i o n of the sex d i f f e r e n c e s in drug and s t e r o i d metabol ism in the STZ-d i a b e t i c animal we hypothes i zed that t h i s may be being mediated in par t by the HCLA es t rogen b i n d i n g p r o t e i n . We thus i n v e s t i g a t e d the e f f e c t s of STZ- induced d i a b e t e s on the HCLA es t rogen b i n d i n g p r o t e i n at t ime p e r i o d s where the changes in AHH a c t i v i t y were ev ident (4 and 10 d a y s ) . We a l s o sought the hormonal c o n t r o l mechanism of the HCLA es t rogen b i n d i n g p r o t e i n in t h i s p a t h o l o g i c a l c o n d i t i o n and 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 s of hormonal replacement in p h y s i o l o g i c a l doses to the S T Z - d i a b e t i c a n i m a l . T e s t o s t e r o n e e n a n t h a t e , protamine z i n c i n s u l i n , 3 ,3 , 5 - t r i i o d o t h y r o n i n e were a d m i n i s t e r e d once a day. Toronto r e g u l a r i n s u l i n was a d m i n i s t e r e d twice a day. With growth hormone four p o s s i b i l i t i e s had to be examined: mean l e v e l s of c i r c u l a t i n g GH need to be m a i n t a i n e d , the peaks of the u l t r a d i e n e p a t t e r n need to be p r e s e n t , the t roughs of the u l t r a d i e n e p a t t e r n need to be p r e s e n t , or both the peaks and t roughs of the u l t r a d i e n e p a t t e r n need to be m a i n t a i n e d . MATERIALS AND METHODS 1) Chemicals and Reagents The f o l l o w i n g chemica ls and reagents were obta ined from Sigma Chemical Company ( S t . L o u i s , Mo . ) : a c t i v a t e d c h a r c o a l , bovine serum albumin (BSA) , Coomassie b r i l l i a n t b lue G, D L - d i t h i o t h r e i t o l (DTT) , d isodium e thy lened iamine t e t r a a c e t i c a c i d (EDTA), sodium molybdate , s t r e p t o z o t o c i n ( S T Z ) , t e s t o s t e r o n e enantha te , 3 ,3 ' , 5 - 1 r i i o d o - L - t h y r o n i n e ( T 3 ) , 1 7 P - e s t r a d i o l , Trizma® base . [ 6 , 7 - 3 H ( N ) ] -E s t r a d i o l , 40-60 Ci/mmol and B i o f l u o r s c i n t i l l a t i o n c o c k t a i l were obta ined from New England Nuc lear ( B o s t o n , M a . ) . Dextran T-70 was obta ined from Pharmacia F ine Chemicals AB ( U p p s a l a , Sweden). Alzet® osmot ic -min ipumps, Model 2001 were o b t a i n e d from A l z a C o r p o r a t i o n ( P a l o A l t a , C a . ) . Ovine growth hormone (1.5 I.U./mg) was a g i f t of the Nat iona l I n s t i t u t e of D i a b e t e s , D i g e s t i v e , and Kidney D i s e a s e s . Protamine z i n c and Toronto i n s u l i n (Connaught Labs) were purchased at r e t a i l o u t l e t s as was the Tes-tape® ( E l i L i l l y and Company, T o r o n t o , O n t ) . A l l o ther chemica ls and reagents were of a n a l y t i c a l q u a l i t y . 2) Animals Male Wis ta r ra t s weighing 300-350 grams, male C57BL/6 and male DBA/2 mice weighing 20-30 grams were obta ined from Canadian Breed ing Farms ( M o n t r e a l , P . Q . ) . Male guinea p igs weighing 325-375 grams were obta ined from l o c a l s u p p l i e r s 1 6 (U.B .C Animal C a r e ) . Animals were housed in a separa te animal room on Lobund® bedding (Paxton P r o c e s s i n g L t d . , Paxton , I I . ) under c o n t r o l l e d l i g h t (6 a.m. on , 8 p.m. o f f ) and temperature (22°C). They were a l lowed f r e e access to food (Pur ina Labora to ry Chow, Ra ls ton Pur ina of Canada L t d , Woodstock, Ont . ) and tap water ad l i b i t u m . 3) Animal Treatments S t r e p t o z o t o c i n was a d m i n i s t e r e d four or ten days p r i o r to animal use vi a a t a i l ve in i n j e c t i o n of 60 mg/kg in c i t r a t e b u f f e r . C o n t r o l s were i n j e c t e d with v e h i c l e on ly under e ther a n a e s t h e t i c . Before i n j e c t i o n , ra ts were a l l owed a minimum of 3-4 days to e q u i l i b r a t e a f t e r sh ipment . The f o l l o w i n g hormones were i n j e c t e d s . c . once a day: protamine z inc i n s u l i n (PZI) (10 u n i t s / k g ) , t e s t o s t e r o n e enanthate (TES) (1 mg/kg in corn o i l ) , t r i i o d o t h y r o n i n e (Tg) (30 fig/kg in normal s a l i n e at pH 8 . 5 ) . Toronto i n s u l i n was a d m i n i s t e r e d s . c . twice a day (15 u n i t s / k g ) . Ovine growth hormone (GH) was d i s s o l v e d in normal s a l i n e (pH = 8.5) and: 1) a d m i n i s t e r e d s . c . in a dose of 30 M -g/inject ion seven t imes per day at 6:15 a . m . , 10:15 a . m . , 2:15 p . m . , 6:15 p .m. , 9:00 p . m . , 11:40 p . m . , and 2:40 a . m . ; 2) f o r cont inuous i n f u s i o n of GH, Alzet® Model 2001 minipumps were implanted subcutaneous ly in the r o s t r a l h a l f of e ther a n e a s t h e t i z e d r a t s . The GH s o l u t i o n was loaded i n t o the pumps and the pumps were weighed be fore and a f t e r f i l l i n g to ensure complete f i l l i n g . The pumps had a l i f e 1 7 expectancy of 7 days at a pumping ra te of 1.04 uL/hr +_ 0. 06 uL/hr ( e q u i v a l e n t to a dose of 0.02 u n i t s / h r ) . For 1. v. i n j e c t i o n GH was a d m i n i s t e r e d vi a the t a i l ve in in consc ious animals four t imes per day (9:00 a . m . , 1:00 p . m . , 5:00 p . m . , and 9:00 p.m.) in a dose of 30 n g / i n j e c t i o n . A r e l a t e d set of exper iments used the same GH i . v . i n j e c t i o n method and had TES and PZI added to the t reatment regimen in the same i n j e c t i o n p a t t e r n and dose as they were when g iven a l o n e . Rats were cannu la ted under ha lothane a n a e s t h e s i a f o l l o w i n g s tandard s u r g i c a l p r o c e d u r e s . Rats were a n a e s t h e t i z e d with ha lothane and a midabdominal i n c i s i o n made through the sk in and muscle l a y e r s . The vena cava was l o c a t e d and sutures passed underneath and over the area where the cannula was to be p l a c e d . A t r o c a r was pushed through the back muscle bes ide the sp ine and a l lowed to e x i t d o r s a l l y at the neck reg ion to c r e a t e a c a v i t y under the s k i n of the back. The cannula was then p laced in the t r o c a r and taken to the e x i t po in t and f low through the cannula was checked . The other end of the cannula was i n s e r t e d i n t o the vena cava and l o o s e l y t i e d to nearby muscle t i s s u e f o r a n c h o r i n g . T o p i c a l a n t i b i o t i c s were s p r i n k l e d over the wound, and the muscle and s k i n l a y e r s were c a r e f u l l y sutured c l o s e d . L i d o c a i n e s o l u t i o n was a p p l i e d t o p i c a l l y to a l l e v i a t e pain from the s u r g e r y . The animal was removed from the h a l o t h a n e , t reatments s t a r t e d , and p laced in an i n d i v i d u a l cage f o r recovery and u s e . 1 8 4) High c a p a c i t y , low a f f i n i t y p r o t e i n b i n d i n g assay Rats and mice were stunned with a blow to the head and k i l l e d by d e c a p i t a t i o n . Guinea p igs were k i l l e d v i a c e r v i c a l d i s l o c a t i o n f o l l o w e d by d e c a p i t a t i o n . L i v e r s were per fused v i a the p o r t a l ve in with i c e co ld TED b u f f e r , c o n t a i n i n g 50mM T r i s , 1.5mM EDTA, 0.5mM DTT, 20mM sodium m o l y b d a t e , and 10% g l y c e r o l , pH 7.5 at 4°C. The l i v e r s were then e x c i s e d i n t o TED b u f f e r , b l o t t e d dry and the weight r e c o r d e d . Tes-tape® was used to t e s t f o r g l y c o s u r i a at the t ime of k i l l i n g to con f i rm the presence of d i a b e t e s . L i v e r s were then minced and homogenized (1:10 w/v) in TED b u f f e r us ing a P o t t e r - E l v e h j e m t i s s u e homogenizer . A f t e r homogenizat ion samples were c e n t r i f u g e d at 10,000g f o r 10 minutes at 4°C us ing an I n t e r n a t i o n a l Equipment Company (Needham H t s . , Ma.) Model B-20 r e f r i g e r a t e d c e n t r i f u g e . The r e s u l t i n g supernatant was c e n t r i f u g e d at 100,000g f o r 30 minutes at 4°C (to o b t a i n the c y t o s o l i c f r a c t i o n ) us ing a Beckman L5-50 u l t r a c e n t r i f u g e with a type 65 f i x e d angle r o t o r (Beckman Instruments C o r p . , Palo A l t o C a . ) . The supernatant was removed and d i l u t e d with TED b u f f e r to a p r o t e i n c o n c e n t r a t i o n of 1-3 mg/ml determined v i a the B r a d f o r d p r o t e i n assay method ( B r a d f o r d , 1976) and p laced on i c e . The b i n d i n g c h a r a c t e r i s t i c s of e s t r a d i o l were determined by Scatchard (1949) a n a l y s i s of c o m p e t i t i v e b i n d i n g c u r v e s . The i n c u b a t i o n mixture conta ined 500 ul c y t o s o l , 10 uL [ 3 H ] - e s t r a d i o l (10 Ci/mmol) 10-200 nM ( in 1 9 abso lu te e t h a n o l ) in the presence or absence of a 100 f o l d excess of u n l a b e l l e d e s t r a d i o l . Incubat ions were c a r r i e d out f o r 90 minutes at 4°C. The r e a c t i o n was te rminated and bound s t e r o i d was s e p a r a t e d from f r e e by the a d d i t i o n of 500 liL of dextran T-70 (0.05% w/v) coated charcoa l (0.5% w/v) (DCC) in TED b u f f e r , as prepared p r e v i o u s l y minus the molybdate . F o l l o w i n g the a d d i t i o n of DCC the samples were mixed and DCC sedimented by c e n t r i f u g a t i o n at 150Og f o r 10 minutes at 4°C us ing a Beckman J6-B r e f r i g e r a t e d c e n t r i f u g e with a type JS 4.2 r o t o r . The r e s u l t i n g supernatants were sampled (0.5 mL) and mixed with 10 mL B i o f l u o r l i q u i d s c i n t i l l a t i o n c o c k t a i l . Samples were counted f o r bound e s t r a d i o l us ing a S e a r l e Mark III L i q u i d S c i n t i l l a t i o n System, Model 6880 ( S e a r l e A n a l y t i c a l I n c . , Des P l a i n s , I I . ) . The Mark III was i n t e r f a c e d with an Apple Plus II computer so data cou ld be recorded d i r e c t l y to d i s k . A l l p o i n t s were assayed in d u p l i c a t e . The tubes which c o n t a i n e d 3 [ H ] - e s t r a d i o l without compet i to r measured t o t a l b i n d i n g 3 ( T B ) , wh i le the tubes with both [ H j - e s t r a d i o l and c o m p e t i t o r represented the n o n - s p e c i f i c b i n d i n g (NSB) p r e s e n t . A n a l y s i s of b i n d i n g data proceeded as f o l l o w s . The 3 counts per minute (cpm) recorded f o r [ H ] - e s t r a d i o l were conver ted to d i s i n t e g r a t i o n s per minute (dpm) based on the percent e f f i c i e n c y of the i n s t r u m e n t . E f f i c i e n c y f o r t r i t i u m was 47%. Thus dpms were c a l c u l a t e d on the b a s i s of e f f i c i e n c y , which was determined f o r each sample from 2 0 p r e v i o u s l y prepared quench s t a n d a r d s . The dpms were 12 c o n v e r t e d to C u r i e s on the b a s i s t h a t 1 Ci = 2.2x10 dpm. The s p e c i f i c a c t i v i t i e s of the l i g a n d s were employed to c a l c u l a t e the p icomoles of b i n d i n g which was normal i zed f o r p r o t e i n c o n t e n t . Then the NSB tube va lues were s u b t r a c t e d from the TB tube va lues to g i ve the s p e c i f i c b i n d i n g (SB) at any p o i n t . D u p l i c a t e s were then a v e r a g e d . The t o t a l l i g a n d ( t o t a l counts) added, l e s s the SB, r e p r e s e n t s the f r e e c o n c e n t r a t i o n (F) used in the c a l c u l a t i o n of s p e c i f i c bound over f r e e ( B / F ) . In cases where d i sp lacement was observed the B/F was graphed as a f u n c t i o n of SB to y i e l d a c u r v e - l i n e a r p l o t with a negat i ve s lope (Sca tchard p l o t ) . The i n v e r s e of the s lope of the l i n e a r p o r t i o n of the p l o t represented the d i s s o c i a t i o n constant ( K d ) , and the x - i n t e r c e p t the u n c o r r e c t e d measure of c a p a c i t y . (The numbers f o r the Scatchard p l o t were obta ined by s u b j e c t i n g the data to computer a n a l y s i s ( Input/Ca l c programme by Sunahara and Fawzi) and by c o n f i r m i n g the r e s u l t s by hand) . 5) Dete rminat ion of P r o t e i n Content P r o t e i n c o n c e n t r a t i o n was determined by the method of B rad ford (1976) . Using BSA as a s t a n d a r d , the assay i s l i n e a r f o r p r o t e i n c o n c e n t r a t i o n s from 0.1 - 0.5 mg/mL. A P e r k i n - E l m e r Model 124 double beam spect rophotometer was used to assess p r o t e i n c o n c e n t r a t i o n . Samples were read 5 minutes a f t e r the a d d i t i o n of 5 mL Brad ford reagent which c o n t a i n e d : 100 mg C o o m a s s i e - b r i 1 1 i a n t b l u e - G ; 50 mL 2 1 a b s o l u t e e t h a n o l ; 100 mL 85% phosphor i c a c i d ; d i l u t e d to IL and f i l t e r e d us ing a Buchner s u c t i o n a p p a r a t u s . 6) S t a t i s t i c a l A n a l y s i s D i f f e r e n c e s were cons ide red s i g n i f i c a n t from c o n t r o l at p<0.05 us ing ANOVA and the Newman-Keuls m u l t i p l e range t e s t . RESULTS 1) E f f e c t of p o s s i b l e blood contamina t ion E a r l y in our s t u d i e s the sugges t ion tha t b lood con tamina t ion cou ld produce a r t i f a c t u a l r e s u l t s was made ( P o e l l i n g e r et al . , 1 9 8 3 ) . To d e t e r m i n e whether t h e r e was a b lood-borne component which may i n t e r f e r e with our a s s a y , v a r i o u s p e r f u s i o n volumes were used to f l u s h the blood out i n s i t u . As w e l l , whole r a t b l o o d ( t a k e n from the a n i m a l s at t ime of d e c a p i t a t i o n ) was assayed f o r the presence of any component which cou ld bind e s t r a d i o l in a s p e c i f i c and s a t u r a b l e manner. As shown in Table I and F i g u r e 1 t h e r e were no s i g n i f i c a n t d i f f e r e n c e s in the apparent b i n d i n g c a p a c i t y or Kd in l i v e r s per fused with 10 or 100 mL of TED b u f f e r . It can a l s o be seen that whole b lood conta ined no agent capable of b i n d i n g e s t r a d i o l . 2) E f f e c t of STZ- induced d i a b e t e s The e f f e c t of a s i n g l e i n j e c t i o n of 60 mg/kg of STZ upon the b i n d i n g c a p a c i t y of the HCLA b i n d i n g p r o t e i n i s shown in Table II and F i g u r e 2. The b i n d i n g c a p a c i t y i s decreased by 50-80% at both the 4 day and 10 day time p o i n t s . The apparent Kd i s not a f f e c t e d by the d i a b e t i c s t a t e . D iabetes was conf i rmed by the presence of g l y c o s u r i a , 1/2% or g r e a t e r . 2 3 Table I EFFECT OF LIVER PERFUSION VOLUME The e f f e c t of v a r y i n g the l i v e r p e r f u s i o n volume was determined in c i t r a t e i n j e c t e d c o n t r o l animals ( c i t r a t e b u f f e r pH 4 . 5 ) . Rat b lood was assayed f o r the presence of any component which was capable of b i n d i n g the l i g a n d in a s p e c i f i c and s a t u r a b l e manner. Incubat ions were c a r r i e d out as d e s c r i b e d in the methods s e c t i o n . B ind ing k i n e t i c s were determined by Scatchard a n a l y s i s . Data were expressed as mean+S.E.M. Numbers in b racke ts denote the number of animals t e s t e d . P e r f u s i o n Apparent B ind ing K i n e t i c s Volume Kd (10" 7 M) Bmax (pmol/mg) 10 mL 1.08+0.34 14.83+3.97 (11) 100 mL 1.41+0.11 17.91+4.82 (8) Rat b lood no d e t e c t a b l e b i n d i n g * (2) * denotes no d e t e c t a b l e d i s p l a c e a b l e and s a t u r a b l e b i n d i n g F i g . 1 E f f e c t of v a r y i n g the l i v e r p e r f u s i o n volume. D i f f e r e n t volumes (10 mL© -o , 100 m L e a ) of TED b u f f e r were p e r f u s e d through the vena cava b e f o r e removal of the l i v e r . Incubat ions were c a r r i e d out a c c o r d i n g to the Methods s e c t i o n . Graphs shown are of a s i n g l e exper iment and are r e p r e s e n t a t i v e of the r e s u l t s f ound . Table II EFFECT OF STREPTOZOTOCIN-INDUCED DIABETES AND INSULIN REPLACEMENT ON THE MALE RAT HEPATIC HCLA BINDING PROTEIN A d u l t male ra t s were rendered d i a b e t i c by a s i n g l e i n j e c t i o n of s t r e p t o z o t o c i n (STZ) (60 mg/kg) in c i t r a t e b u f f e r (pH 4.5) under e ther a n a e s t h e t i c ( c o n t r o l s were i n j e c t e d with v e h i c l e o n l y ) . Protamine z i n c i n s u l i n (PZI) was a d m i n i s t e r e d in a dose of 10 un i t s/kg s . c . once a day, Toronto i n s u l i n ( T o . I ) was g iven in a dose of 15 un i t s/kg s . c . twice a day. Incubat ions were c a r r i e d out as d e s c r i b e d in the Methods s e c t i o n . B ind ing k i n e t i c s were determined by Sca tchard a n a l y s i s . Data were expressed as mean +_ S . E . M . Numbers in b r a c k e t s denote number of animals t e s t e d . Treatment Apparent B ind ing K i n e t i c s Kd (10"'M) Bmax (pmol/mg) Cont ro l 1.08+0.34 14.83+3.97 (11) 4 day STZ 0.82+0.18 4.82+0.95* (8) 4 day STZ + PZI 0.79+0.19 3.41+0.99* (5) 4 day STZ +To. I 0.57+0.06 1.41+0.38* (7) 10 day STZ 0.51+0.26 4.37+1.59* (5) 10 day STZ + PZI 1.08+0.18 5.72 1.23* (5) * denotes s i g n i f i c a n t d i f f e r e n c e compared to c o n t r o l (p<0.05) a c c o r d i n g to ANOVA and the Newman-Keul range t e s t 2 7 F i g . 2 E f f e c t of 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 e s on the l e v e l s of the HCLA b ind ing p r o t e i n of rat l i v e r . Adu l t male ra ts were rendered d i a b e t i c by a s i n g l e t a i l ve in i n j e c t i o n of s t r e p t o z o t o c i n (60 mg/kg) in c i t r a t e b u f f e r (pH 4.5) under e ther a n a e s t h e s i a ; c o n t r o l s (o o) were i n j e c t e d with b u f f e r o n l y . Animals were l e f t f o r 4 (•- o) or 10 (A A) days be fore use . Incubat ions were c a r r i e d out as d e s c r i b e d in the Methods s e c t i o n . Graphs shown are of a single" experiment and are r e p r e s e n t a t i v e of the r e s u l t s f ound . 2 9 3) E f f e c t of i n s u l i n rep lacement in the S T Z - d i a b e t i c animal The e f f e c t of protamine z i n c i n s u l i n t reatment in a dose of 10 un i t s/kg s . c . once a day can be seen in Table II and F i g u r e 3. Th is t reatment regimen was i n e f f e c t i v e in r e s t o r i n g the l e v e l s of the HCLA b i n d i n g p r o t e i n i n both the 4 day and 10 day d i a b e t i c a n i m a l s . The apparent b i n d i n g c a p a c i t y remained depressed to l e v e l s 50-80% below of those seen in c o n t r o l animals and the Kd was u n a f f e c t e d . Protamine z i n c i n s u l i n was e f f e c t i v e in c o n t r o l l i n g the d i a b e t i c s t a t e as ev idenced by the lack of g l y c o s u r i a i n the t r e a t e d a n i m a l s . S ince there were no d i f f e r e n c e s seen between the 4 day and 10 day time p o i n t s the 4 day model was used f o r the res t of the exper iments . Toronto r e g u l a r i n s u l i n was t e s t e d to see i f i t cou ld cause the r e v e r s a l of the d e c r e a s e in b i n d i n g c a p a c i t y of the HCLA b i n d i n g p r o t e i n due to a repor t in the l i t e r a t u r e . O r t i z - C a r o et a l . (1984) found that Toronto r e g u l a r i n s u l i n was e f f e c t i v e in reduc ing some of the t h y r o i d d y s f u n c t i o n seen in the S T Z - d i a b e t i c animal when g iven in a regimen of 15 u n i t s / k g s . c . twice a day, whereas protamine z i n c i n s u l i n once a day had been i n e f f e c t i v e . The lack of r e s t o r a t i o n of HCLA es t rogen b i n d i n g p r o t e i n l e v e l s with t h i s t reatment can be seen in Table II and F i g u r e 3. With respec t to the HCLA b i n d i n g p r o t e i n i t would appear that the type of i n s u l i n and i t s dosage d id not make a d i f f e r e n c e as we were again unable to r e s t o r e the reduced b i n d i n g c a p a c i t y . The d i a b e t i c s t a t e was being c o n t r o l l e d as 3 0 F i g . 3 E f f e c t of i n s u l i n on the l e v e l of the h e p a t i c HCLA b i n d i n g p r o t e i n 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 r a t s . A d u l t male rats were rendered d i a b e t i c by a s i n g l e t a i l ve in i n j e c t i o n of s t r e p t o z o t o c i n (60 mg/kg) i n c i t r a t e b u f f e r (pH 4.5) under e ther a n a e s t h e s i a ; c o n t r o l s fp CD) were i n j e c t e d with b u f f e r o n l y . Protamine z i n c i n s u l i n was g iven in a dose of 10 un i t s/kg s . c . once a day. Animals were t r e a t e d f o r 4 (• •) or 10 ( A A ) days be fore u s e . Toronto r e g u l a r i n s u l i n was g iven in a dose of 15 u n i t s / k g s . c . twice a day f o r 4 days (v v) be fore u s e . Incubat ions were c a r r i e d out as d e s c r i b e d in the Methods s e c t i o n . Graphs shown are of a s i n g l e exper iment and are r e p r e s e n t a t i v e of the r e s u l t s f ound . 3 2 ev idenced by the lack of g l y c o s u r i a . 4) E f f e c t of subcutaneous i n j e c t i o n s The e f f e c t of subcutaneous i n j e c t i o n of v e h i c l e s was d e t e r m i n e d i n c i t r a t e b u f f e r (pH=4.5) i n j e c t e d ( v i a a t a i l ve in under e ther a n e a s t h e s i a ) c o n t r o l a n i m a l s . Normal s a l i n e was i n j e c t e d s . c . at a volume of 0.3 mL once a day , or corn o i l was i n j e c t e d s . c . at a volume of 0.5 mL once a day. The r e s u l t s can be seen in Table I I I . It i s apparent that t h e r e i s no change in e i t h e r the apparent b i n d i n g c a p a c i t y or Kd between i n j e c t e d or n o n - i n j e c t e d c o n t r o l ani ma ls . 5) E f f e c t of hormone replacement a) T e s t o s t e r o n e enanthate Due to the decrease in serum t e s t o s t e r o n e seen in the d i a b e t i c male and the r e s t o r a t i v e e f f e c t of t e s t o s t e r o n e on the HCLA b i n d i n g p r o t e i n in the gonadectomized male , we i n v e s t i g a t e d the e f f e c t of t e s t o s t e r o n e replacement in the d i a b e t i c male. The e f f e c t of t e s t o s t e r o n e enanthate at a dose of 1 mg/kg can be seen in Tab le IV and F i g u r e 4. It i s observed that t h i s dose , which i s s u f f i c i e n t to r e s t o r e normal plasma t e s t o s t e r o n e l e v e l s , (Sunahara , 1984) i s unab le to r e s t o r e the decreased b i n d i n g c a p a c i t y seen i n the STZ- induced d i a b e t i c a n i m a l . The Kd remains the same as tha t seen in the c o n t r o l a n i m a l s . The d i a b e t i c animals 3 3 Table III EFFECT OF SALINE AND CORN OIL S . C . INJECTIONS IN CONTROL ANIMALS A d u l t male ra t s were i n j e c t e d with 0.3 ml c i t r a t e b u f f e r (pH 4.5) under e ther a n e a s t h e t i c . Normal s a l i n e was i n j e c t e d s . c . at a volume of 0.3 ml once a day. Corn o i l was i n j e c t e d at a volume of 0.5 ml once a day . I n c u b a t i o n s were c a r r i e d out as d e s c r i b e d in the Methods s e c t i o n . B i n d i n g k i n e t i c s were determined by Scatchard a n a l y s i s . Data were expressed as mean _+ S . E . M . Numbers in b racke ts denote number of animals t e s t e d . Treatment Apparent B ind ing K i n e t i c s Kd (10 ) Bmax (pmol/mg) S a l i n e s . c . 1.01+0.01 14.71+1.53 (4) Corn o i l s . c . 0.90+0.01 12.68+2.83 (4) Cont ro l 1.08+0.34 14.83+3.97 (11) t r e a t e d with t e s t o s t e r o n e s t i l l showed s igns of g l y c o s u r i a at the time of k i l l i n g . b) 3 , 3 , 5 - t r i i o d o t h y r o n i n e S ince there are a l t e r a t i o n s in t h y r o i d hormone l e v e l s in the d i a b e t i c a n i m a l , we i n v e s t i g a t e d the replacement of t r i i o d o t h y r o n i n e . The e f f e c t of a d m i n i s t e r i n g t r i i o d o t h y r o n i n e at a dose of 30 ng/kg can be seen in Tab le IV and F i g u r e 4. Th is dosage i s e f f e c t i v e in r e s t o r i n g the t h y r o i d to normal s t a t u s ( T a h i l i a n a , 1983). It can be seen that the replacement of t r i i o d o t h y r o n i n e in the d i a b e t i c animal i s unable to r e s t o r e the decreased b i n d i n g c a p a c i t y but the Kd was u n a l t e r e d from the c o n t r o l v a l u e s . The d i a b e t i c animals t r e a t e d with t r i i o d o t h y r o n i n e t e s t e d p o s t i v e f o r g l y c o s u r i a at the t ime of k i l l i n g . c) Growth hormone The e f f e c t of replacement of growth hormone can be seen in Tab le IV and F i g u r e 5. C a l c u l a t i n g from known blood l e v e l s (Tannenbaum and M a r t i n , 1976) two t reatment regimens were c a r r i e d out with growth hormone: i ) replacement v i a cont inuous i n f u s i o n with the use of minipumps; i i ) replacement vi a a seven t imes per day s . c . i n j e c t i o n s c h e d u l e . Ne i the r of these t reatment regimens (done to r e s t o r e a b s o l u t e l e v e l s of growth hormone and to d u p l i c a t e the n a t u r a l male r e l e a s e p a t t e r n r e s p e c t i v e l y ) were ab le to r e s t o r e the decreased b i n d i n g c a p a c i t y of the HCLA b i n d i n g 3 5 Table IV EFFECT OF HORMONE REPLACEMENT ON THE RAT HEPATIC HCLA BINDING PROTEIN IN 4-DAY DIABETIC ANIMALS A d u l t male ra t s were rendered d i a b e t i c by a s i n g l e i n j e c t i o n of s t r e p t o z o t o c i n • ( S T Z ) (60 mg/kg) in c i t r a t e b u f f e r (pH 4.5) under e ther a n a e s t h e s i a . T e s t o s t e r o n e enanthate was g iven in a dose of 1 mg/kg in corn o i l once a day. T r i i o d o t h y r o n i n e was g iven in a dose of 30 ug/day s . c . in normal s a l i n e . The animals which had osmotic minipumps implanted were given a dose of ovine growth hormone (1.5 I.U./mg) at 0.02 u n i t s / h o u r f o r four days . The dose of ov ine growth hormone g iven s . c . was 30 u g / i n j e c t i o n seven t imes a day. Incubat ions were c a r r i e d out as d e s c r i b e d in the Methods s e c t i o n . B ind ing k i n e t i c s were determined by Scatchard a n a l y s i s . Data were expressed as mean +_ S . E . M . Numbers in b racke ts denote number of animals t e s t e d . Treatment Apparent B i n d i n g K i n e t i c s Kd (10~'M) Bmax (pmol/mg) Cont ro l 1.08+0.34 14.83+3.97 (11) 4 day STZ 0.82+0.18 4.82+0.95* (8) 4 day STZ + t e s t o s t e r o n e enanthate 0.56+0.10 3.65+0.82* (5) 4 day STZ +t r i i odothyron i ne 0.40+0.10 1.83+0.40* (5) 4 day STZ +growth hormone (minipump) 0.56^0.11 1.81+0.22* (5) 4 day STZ +growth hormone ( s . c . ) 0.52+0.06 1.54+0.28* (7) * denotes s i g n i f i c a n t d i f f e r e n c e compared to c o n t r o l (p<0.05) a c c o r d i n g to ANOVA and the Newman-Keuls range t e s t 3 6 F i g . 4 E f f e c t of t e s t o s t e r o n e and 3 ,3 , 5 - t r i i o d o t h y r o n i n e replacement in 4 day 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 animals on the l e v e l of the HCLA b i n d i n g p r o t e i n of rat l i v e r . Adu l t male ra t s were made d i a b e t i c by a s i n g l e t a i l ve in i n j e c t i o n of s t r e p t o z o t o c i n (60 mg/kg) in c i t r a t e b u f f e r (pH 4.5) under e ther a n a e s t h e s i a ; c o n t r o l s (o o) were i n j e c t e d with b u f f e r o n l y . Animals were t r e a t e d with t e s t o s t e r o n e enanthate at a dose of 1 mg/kg in corn o i l s . c . once a day (b • ) , or t r i i o d o t h y r o n i n e at a dose of 30 ug/kg in normal s a l i n e s . c . once a day (A .—-A) . Incubat ions were c a r r i e d out as d e s c r i b e d in the Methods s e c t i o n . Graphs shown are of a s i n g l e experiment and are r e p r e s e n t a t i v e of the r e s u l t s found . 0 2 4 6 B 10 S p e c i f i c B i n d i n g (ptnol/mg) 3 8 F ig . 5 E f f e c t of ov ine growth hormone (1.5 I.U./mg) replacement on the h e p a t i c HCLA b i n d i n g p r o t e i n in 4 day 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 . Adu l t male ra ts were rendered d i a b e t i c by a s i n g l e t a i l ve in i n j e c t i o n of s t r e p t o z o t o c i n (60 mg/kg) in c i t r a t e b u f f e r (pH 4.5) under e ther a n a e s t h e s i a ; c o n t r o l s (o were i n j e c t e d with b u f f e r o n l y . Growth hormone was d i s s o l v e d in b a s i c (pH 8 .5) s a l i n e and admin i s te red by e i t h e r osmotic minipump (• •) imp lan ted in the r o s t r a l p o r t i o n of the r a t ' s back and g iven a dose of 0.02 u n i t s / h r , or i n j e c t e d s . c . (A A) at a dose of 30 u g / i n j e c t i o n seven t imes a day. Animals were t r e a t e d f o r 4 days be fore u s e . Incubat ions were c a r r i e d out as d e s c r i b e d in the Methods s e c t i o n . Graphs shown are of a s i n g l e experiment and are r e p r e s e n t a t i v e of the r e s u l t s f o u n d . 0 2 4 6 8 S p e c i f i c B i n d i n g (pmol/mg) 4 0 p r o t e i n in the d i a b e t i c r a t . A g a i n , the Kd va lue was u n a l t e r e d from the va lue seen in c o n t r o l a n i m a l s , and the animals t e s t e d p o s i t i v e f o r g l y c o s u r i a . 6) E f f e c t of i n t ravenous growth hormone a d m i n i s t r a t i o n a) Vena cava c a n n u l a t i o n The e f f e c t of vena cava c a n n u l a t i o n and the subsequent a d m i n i s t r a t i o n of growth hormone to the S T Z - d i a b e t i c animal can be seen i n Tabel V. T h i s was under taken to more c l o s e l y d u p l i c a t e the normal p h y s i o l o g i c l e v e l seen in the blood ( i . e . to get marked blood p e a k s ) . There was no r e s t o r a t i o n of the decreased b i n d i n g c a p a c i t y and t h i s t reatment a c t u a l l y gave the lowest l e v e l s of the HCLA b i n d i n g p r o t e i n tha t we o b s e r v e d ; the Kd remained u n a l t e r e d from the c o n t r o l v a l u e . Because of t h i s very low b i n d i n g c a p a c i t y concern was r a i s e d over the p o t e n t i a l h e p a t o t o x i c i y of ha lothane as an a n a e s t h e t i c and a c o n t r o l exper iment was done to compare the e f f e c t s of ha lothane versus e ther as the s u r g i c a l a n a e s t h e t i c . The r e s u l t s of t h i s can be seen in Table V. There were no apparent d i f f e r e n c e s in the b i n d i n g c a p a c i t y va lues obta ined with e i t h e r ha lothane or e ther a n a e s t h e s i a , but these va lues were s i g n i f i c a n t l y lower than the va lues obta ined from c o n t r o l animals which had not been s u r g i c a l l y m a n i p u l a t e d ; the Kd va lues were the same in a l l th ree c a n n u l a t e d groups and were not d i f f e r e n t from the c o n t r o l Kd v a l u e s . The low number of animals used to o b t a i n data p r e c l u d e d s t a t i s t i c a l m a n i p u l a t i o n of the f i g u r e s . La rger 4 1 Table V EFFECT OF VENA CAVA CANNULATION ON THE THE RAT HEPATIC HCLA BINDING PROTEIN Adu l t male ra ts were cannu la ted as d e s c r i b e d in the Methods s e c t i o n under e i t h e r e ther or ha lothane a n a e s t h e s i a . One group was rendered d i a b e t i c by a s i n g l e i n j e c t i o n of s t r e p t o z o t o c i n (STZ) (60 mg/kg) in c i t r a t e b u f f e r (pH 4 .5) w h i l e under the o p e r a t i v e a n a e s t h e t i c ( c o n t r o l s were i n j e c t e d with b u f f e r o n l y ) . Ovine growth hormone (1.5 I.U./mg) was given in a dose of 30 u g / i n j e c t i o n s . c . seven time a day. B ind ing k i n e t i c s were determined by Sca tchard a n a l y s i s . Numbers in b racke ts denote the number of animals t e s t e d . Data were presented as the mean +_ S . E . M . Incubat ions were c a r r i e d out as d e s c r i b e d in the Methods s e c t i o n . C o n d i t i o n s of Apparent B i n d i n g K i n e t i c s C a n n u l a t i on Kd ( 1 0 ' ' M ) Bmax ( p m o l / m g ) e ther a n a e s t h e t i c 0.53+0.17 2.85+1.67 (3) ha lothane a n a e s t h e t i c 0.59+0.13 4.25+2.44 (3) ha lothane a n a e s t h e t i c +STZ tgrowth hormone 0.46+0.20 0.51+0.19 (2) numbers of animals were u s e d , but because of the i n t o l e r a n c e of the rat to the c a n n u l a , as demonstrated by i t s p u l l i n g the cannula o u t , chewing the i n j e c t i o n end down to where i t was u n u s a b l e , and the problem of b lood c l o t s p lugg ing the cannula i n s i d e the a n i m a l , the f i n a l numbers were low. b) T a i l ve in a d m i n i s t r a t i o n in the consc ious animal The e f f e c t of t a i l ve in a d m i n i s t r a t i o n of growth hormone i s shown in Table VI and F i g u r e 6. A l l t reatment groups showed a decrease in the b i n d i n g c a p a c i t y of the HCLA b i n d i n g p r o t e i n compared to the unt rea ted ( i . e . u n i n j e c t e d ) c o n t r o l s , with no d i f f e r e n c e s in the Kd v a l u e s . Th i s d e c r e a s e in b i n d i n g c a p a c i t y was a l s o e v i d e n t in the v e h i c l e i n j e c t e d c o n t r o l an imals which were only be ing i n j e c t e d with s a l i n e in the t a i l v e i n . These animals showed an approx imate ly 50% decrease in b ind ing c a p a c i t y compared to n o n - i n j e c t e d c o n t r o l s , i . e . from 10-15 pmol/mg p r o t e i n to 5 pmol/mg p r o t e i n . The STZ i n s i d e c o n t r o l s ( i n j e c t e d with s a l i n e ) showed a f u r t h e r 50% d e c r e a s e below t h i s l e v e l , i . e . to 2 pmol/mg p r o t e i n , as d id the growth hormone t r e a t e d d i a b e t i c s . A second t reatment group in which t e s t o s t e r o n e enanthate and protamine z i n c i n s u l i n were a l s o g i v e n , in doses d e s c r i b e d p r e v i o u s l y , in a d d i t i o n to the i . v . growth hormone showed a decrease s i m i l a r to tha t seen with the growth hormone alone and the S T Z - d i a b e t i c animal i n j e c t e d with s a l i n e . A l l the STZ i n j e c t e d animals except those t r e a t e d with i n s u l i n showed g l y c o s u r i a at the time of Table VI EFFECT OF HORMONE REPLACEMENT BY TAIL VEIN INJECTION IN CONSCIOUS 4-DAY DIABETIC ANIMALS ON THE RAT HEPATIC HCLA BINDING PROTEIN Adu l t male r a t s were rendered d i a b e t i c by a s i n g l e i n j e c t i o n of s t r e p t o z o t o c i n (STZ) (60 mg/kg) in c i t r a t e b u f f e r (pH 4.5) under e ther a n a e s t h e s i a ( c o n t r o l s were i n j e c t e d with b u f f e r o n l y ) . Ovine growth hormone (1.5 I.U./mg) was i n j e c t e d i . v . at a dose of 30 u g / i n j e c t i o n four t imes a day in normal s a l i n e ( v e h i c l e i n j e c t e d c o n t r o l s and v e h i c l e i n j e c t e d d i a b e t i c c o n t r o l s were i n j e c t e d with s a l i n e o n l y ) . T e s t o s t e r o n e enanthate was g iven at a dose of 1 mg/kg s . c . in corn o i l once a day. Protamine z i n c i n s u l i n (PZI) was g i v e n in a dose of 10 u n i t s / k g s . c . once a d a y . I n c u b a t i o n s were c a r r i e d out as d e s c r i b e d in the Methods s e c t i o n . B i n d i n g k i n e t i c s were determined by Sca tchard a n a l y s i s . Data were expressed as mean _+ S . E . M . Numbers in b racke ts denote number of animals t e s t e d . Treatment Apparent B ind ing K i n e t i c s  Kd (10" 7 M) Bmax (pmol/mg) Cont ro l 1.08+0.34 14.83+3.97 (11) V e h i c l e i n j e c t e d Con t ro l 0.66+0.11 4.05+0.47* (4) V e h i c l e i n j e c t e d 4 day STZ 4 day STZ +growth hormone 0.36+0.03 2.07+0.37** (4) 0.43+0.04 2.25+0.34** (4) 4 day STZ +growth hormone + t e s t o s t e r o n e enanthate + PZI 0.54+0.18 2.37+0.37** (4) * denotes a s i g n i f i c a n t d i f f e r e n c e from c o n t r o l (p<0.05) a c c o r d i n g to ANOVA and the Newman-Keuls range t e s t ** denotes a s i g n i f i c a n t d i f f e r e n c e from c o n t r o l and v e h i c l e i n j e c t e d c o n t r o l (p<0.05) a c c o r d i n g to ANOVA and the Newman-Keul s range t e s t 4 4 F i g . 6 E f f e c t of t a i l ve in i n j e c t i o n s in consc ious 4 day 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 ra t s on the h e p a t i c HCLA b i n d i n g p r o t e i n . Adu l t male ra t s were made d i a b e t i c by a s i n g l e t a i l ve in i n j e c t i o n of s t r e p t o z o t o c i n (60 mg/kg) in c i t r a t e b u f f e r (pH 4.5) under e ther a n a e s t h e s i a ; c o n t r o l s (•———•) were i n j e c t e d with b u f f e r o n l y . Cont ro l (D— o) and one group of d i a b e t i c s (• •) were i n j e c t e d in a t a i l ve in with s a l i n e a lone 4 t imes a day f o r 4 days before u s e . Another group of d i a b e t i c animals was t r e a t e d with growth hormone d i s s o l v e d in b a s i c (pH 8.5) s a l i n e at a dose of 30 u g / i n j e c t i o n 4 t imes a day in a t a i l ve in f o r 4 days be fore use (A A). The l a s t group of d i a b e t i c s was t r e a t e d with growth hormone in b a s i c (pH 8.5) s a l i n e at a dose of 30 u g / i n j e c t i o n 4 t imes a day in a t a i l ve in , p lus t e s t o s t e r o n e enanthate at a dose of 1 mg/kg i n corn o i l s . c . once a day , p lus protamine z i n c i n s u l i n at a dose of 10 u n i t s / k g s . c . once a day f o r 4 days be fore use (v v) . Incubat ions were c a r r i e d out as d e s c r i b e d in the Methods s e c t i o n . Graphs shown are of a s i n g l e experiment and are r e p r e s e n t a t i v e of the r e s u l t s f o u n d . 4 5 0 2 4 6 8 S p e c i f i c B i n d i n g (pmol/mg) k i l l i n g . Thus , none of the t reatments i n v o l v e d in t h i s study were able to reverse the decrease in b i n d i n g c a p a c i t y seen in the S T Z - d i a b e t i c a n i m a l . The d i f f e r e n c e between the b i n d i n g c a p a c i t i e s of the v e h i c l e i n j e c t e d c o n t r o l s , and the v e h i c l e i n j e c t e d STZ c o n t r o l s , growth hormone t r e a t e d , and growth hormone + t e s t o s t e r o n e + i n s u l i n t r e a t e d animals were s i g n i f i c a n t , i n d i c a t i n g that there i s s t i l l a d i f f e r e n c e between the n o n - d i a b e t i c animals and the d i a b e t i c animals That i s , even though t h i s p r o t o c o l i s caus ing an e f f e c t on the a n i m a l , probab ly through the s t r e s s of hand l ing and i n j e c t i o n , the d i a b e t i c e f f e c t i s a l s o o c c u r i n g in p a r a l l e l . 7) Other s p e c i e s examined S e v e r a l o ther s p e c i e s were examined f o r the p resence of the hepat i c HCLA b i n d i n g p r o t e i n . The r e s u l t s of these exper iments can be seen in Tab le V I I . None of the other s p e c i e s examined showed any ev idence of the HCLA b i n d i n g p r o t e i n in t h e i r l i v e r c y t o s o l . 8) S t a b i l i t y s t u d i e s It had been suggested that the d i f f e r e n c e s in b i n d i n g c a p a c i t y seen between the c o n t r o l and S T Z - d i a b e t i c animal may be due to d i f f e r e n c e s in the s t a b i l i t y of the p r o t e i n dur ing the i n c u b a t i o n p e r i o d . Th is was examined and the r e s u l t s can be seen in Tab le V I I I . Both the c o n t r o l and S T Z - d i a b e t i c c y t o s o l s demonstrated a s t a b i l i t y of b i n d i n g c a p a c i t y and Kd up to the l onges t t ime po in t measured ( i n t h i s case 3 h o u r s ) . The observed d i f f e r e n c e in the b i n d i n g c a p a c i t y was ev ident ( i . e . c o n t r o l s being approx imate ly 3 t imes h igher than S T Z - d i a b e t i c s ) and the Kd va lues between the two groups were the same. 4 8 Table VII OTHER MODELS EXAMINED FOR THE PRESENCE OF THE HCLA BINDING PROTEIN Mouse and guinea pig l i v e r was assayed f o r the HCLA b i n d i n g p r o t e i n in the same manner as rat l i v e r . B ind ing k i n e t i c s were determined by Scatchard a n a l y s i s . Numbers in b racke ts denote the number of animals t e s t e d . Incubat ions were c a r r i e d out as d e s c r i b e d in the Methods s e c t i o n . Animal model Apparent B ind ing K i n e t i c s Kd ( 1 0 ' 1M ) Bmax ( p m o l / m g ) C57BL/6 mouse 1i ver no d e t e c t a b l e b i n d i n g * (4 poo ls of 3) DBA/2 mouse l i v e r no d e t e c t a b l e b i n d i n g * (4 pools of 3) guinea p ig l i v e r no d e t e c t a b l e b i n d i n g * (4) * denotes no d e t e c t a b l e d i s p l a c a b l e and s a t u r a b l e b i n d i n g Table VIII STABILITY STUDY OF THE HCLA BINDING PROTEIN IN CONTROL AND 4-DAY DIABETIC ANIMALS Adu l t male ra ts were made d i a b e t i c by a s i n g l e i n j e c t i o n of s t r e p t o z o t o c i n (60 mg/kg) in c i t r a t e b u f f e r (pH 4.5) under e t h e r a n e a s t h e t i c ( c o n t r o l s were i n j e c t e d wi th b u f f e r o n l y ) . Cy toso l was incubated immediate ly (0 h r . ) , or l e f t s i t t i n g on i c e f o r 1 hr be fore use , or l e f t s i t t i n g on i c e 3 h r . b e f o r e u s e . I n c u b a t i o n s were c a r r i e d out as d e s c r i b e d i n the Methods s e c t i o n . B i n d i n g k i n e t i c s were determined by Sca tchard a n a l y s i s . Data were expressed as mean+_S.E.M. Numbers in b rackets denote number of animals t e s t e d . Treatment Apparent B ind ing K i n e t i c s  Kd (10" 7 M) Bmax (pmol/mg) Cont ro l 0 h r . 1 h r . 3 h r . 4 day d i a b e t i c 0 h r . 1 h r . 3 h r . 0.72+0.01 0.70+0.01 0.89+0.02 0.47+0.02 0.60+0.01 0.68+0.01 12.59+0.66 (2) 13.29+0.68 (2) 12.96+0.58 (2) 2.78+1.56 (2) 3.98+0.15 (2) 3.47+0.46 (2) 5 0 DISCUSSION 1) B ind ing k i n e t i c s The r e s u l t s ob ta ined in these exper iments f o r the b i n d i n g k i n e t i c s of the HCLA es t rogen b i n d i n g p r o t e i n of male rat l i v e r (Kd=10~ 7M, b i n d i n g capac i ty=14.83 pmol/mg p r o t e i n ) are s i m i l a r to those repor ted by o ther authors (Eagon et al . , 1980; Warren , 1982; F i n l a y s o n , 1983). The s a t u r a t i o n curves (see Appendix I) i n d i c a t e that s p e c i f i c , s a t u r a b l e b ind ing i s t a k i n g p lace in the assay system used , and r e l i a b l e S c a t c h a r d p l o t s can be generated from the d a t a . S ince there was no e f f e c t of v a r y i n g the l i v e r p e r f u s i o n volume, and no s p e c i f i c b i n d i n g cou ld be de tec ted in rat whole b l o o d , the HCLA b i n d i n g p r o t e i n i s a component of l i v e r c y t o s o l and not the plasma t r a n s p o r t p r o t e i n s such as a lbumin and sex hormone b i n d i n g g l o b u l i n which can a l s o bind s t e r o i d s (Anderson , 1974; S i i t e r i et a l . , 1982). S ince there was no b i n d i n g de tec ted in whole blood there were no b lood borne contaminants i n t e r f e r i n g with the assay system. The lack of n o n - s p e c i f i c b i n d i n g which might lead to a r t i f a c t u a l r e s u l t s had a l s o been demonstrated by F i n l a y s o n (1983) who f a i l e d to de tec t b i n d i n g to bovine serum albumin and rat p lasma. 2) E f f e c t of STZ- induced d i a b e t e s STZ- induced d i a b e t e s caused a d e c r e a s e of 50-8 0% i n the 5 1 b i n d i n g c a p a c i t y of the HCLA b i n d i n g p r o t e i n at both the 4 and 10 day t ime p o i n t s . No change i n the Kd was n o t e d . The l e v e l s of the HCLA p r o t e i n , be ing equal at both t ime p o i n t s , i n d i c a t e t h a t the r e d u c t i o n in b i n d i n g c a p a c i t y occurs e a r l y and i s not sub jec t to r e g e n e r a t i o n w i t h i n t h i s t ime p e r i o d . As can be seen in Appendix I , both the c o n t r o l and d i a b e t i c an ima ls demonstrated s p e c i f i c , s a t u r a b l e b i n d i n g , i n d i c a t i n g tha t r e l i a b l e Scatchard p l o t s cou ld be generated f o r both g roups . The r e s u l t s in Table VIII i n d i c a t e tha t there appears to be no apparent a l t e r a t i o n in the s t a b i l i t y of the p r o t e i n per se as a r e s u l t of the STZ t r e a t m e n t , as seen by i t s remaining as s t a b l e as the c o n t r o l over the time course s t u d i e d (but at i t s reduced c a p a c i t y ) . It could be ques t ioned as to whether the decrease seen in the HCLA b i n d i n g p r o t e i n l e v e l s could be due to a d i r e c t e f f e c t of STZ, u n r e l a t e d to i t s d i a b e t o g e n i c a c t i o n ( i . e . i s there a g e n e r a l i z e d decrease in h e p a t i c p r o t e i n s ) . Th is does not seem to be the c a s e , as Montoya and Herrara (1974) observed an o v e r a l l i n c r e a s e in h e p a t i c p r o t e i n c o n c e n t r a t i o n in the S T Z - d i a b e t i c a n i m a l . Observa t ions by o ther groups (Reinke et a l . , 1978; Stohs et a l . , 1979 ; Past and Cook, 1980; Peng et a l . , 1983; Warren et a l . , 1983) a l s o show that t h e r e i s not a g e n e r a l i z e d d e p r e s s i o n of p r o t e i n s in the d i a b e t i c s t a t e ; some p r o t e i n s are decreased and o ther p r o t e i n s are i n c r e a s e d . A l t e r a t i o n s in the l e v e l s of the a f f e c t e d p r o t e i n s occur in p a r a l l e l in the spontaneous ly d i a b e t i c ra t s and the STZ- induced d i a b e t i c r a t s . More s p e c i f i c a l l y , with regard to the HCLA b i n d i n g p r o t e i n , Warren (1982) us ing a s i m i l a r assay system and examining spontaneous ly d i a b e t i c B .B. ra t s d id not see as marked a change in the b i n d i n g c a p a c i t y us ing S c a t c h a r d a n a l y s i s , but upon the a p p l i c a t i o n of the n o n - l i n e a r curve f i t t i n g computer program (NONLIN UBC) a 60% decrease was noted from c o n t r o l v a l u e s . These exper iments were done with t i s s u e poo ls and on a small number of a n i m a l s . U n f o r t u n a t e l y , we were unable to ob ta in any spontaneous ly d i a b e t i c B.B. r a t s to examine and compare r e s u l t s to those obta ined by Warren to see i f the lack of change seen with Scatchard a n a l y s i s cou ld be due to the i n d i v i d u a l v a r i a t i o n observed in the HCLA b i n d i n g p r o t e i n l e v e l s noted in the animals us ing t h i s assay system ( F i n l a y s o n , 1983). However, t h i s repor t and that of Warren agree in that there i s a general t rend towards reduced c a p a c i t y of the HCLA b i n d i n g p r o t e i n in the d i a b e t i c animal ( r e g a r d l e s s of the e t i o l o g y of the d i s e a s e s t a t e i . e . e i t h e r spontaneous or c h e m i c a l l y induced) i n d i c a t i n g that the r e d u c t i o n i s due to the d i a b e t i c s t a t e and not a n o n s p e c i f i c e f f e c t of the S T Z - t r e a t m e n t . The l ack of any e f f e c t on the Kd in the d i a b e t i c s t a t e was a l s o observed by Warren. Th i s lack of change in the Kd i n d i c a t e s tha t probab ly a l l that i s being a l t e r e d in the d i a b e t i c s i s the number of b i n d i n g s i t e s a v a i l a b l e on the p r o t e i n , the a b s o l u t e amount of the p r o t e i n p roduced , or the amount a v a i l a b l e to the l i g a n d in the c y t o s o l i n a f u n c t i o n a l form. 5 3 3) Hormone replacement a) I n s u l i n I n s u l i n rep lacement ( e i t h e r with protamine z i n c i n s u l i n once per day , or Toronto r e g u l a r i n s u l i n twice per day) at l e v e l s which were e f f e c t i v e in c o n t r o l l i n g g l y c o s u r i a , and which had p r e v i o u s l y been shown to reverse the e f f e c t s of d i a b e t e s on h e p a t i c d r u g / s t e r o i d metabo l i sm, were i n e f f e c t i v e in r e v e r s i n g the decrease in b i n d i n g c a p a c i t y of the HCLA b i n d i n g p r o t e i n . Th is was ev ident at both of the t reatment t ime p o i n t s i n v e s t i g a t e d ( 4 and 10 d a y s ) . In c o n t r a s t , r e s t o r a t i o n of normal d r u g / s t e r o i d metabol ism was ev iden t with 4 days of i n s u l i n t reatment (Warren, 1982) . Th is demostrates that i n s u l i n l e v e l s per se are not a c o n t r o l l i n g f a c t o r in the HCLA b i n d i n g p r o t e i n l e v e l s , and tha t there does not appear to be an i n t e r p l a y between the two. Th is i s in c o n t r a s t to the es t rogen r e c e p t o r system where there i s an i n t e r r e l a t i o n s h i p between i n s u l i n , i n s u l i n r e c e p t o r s , and es t rogen r e c e p t o r l e v e l s ( S h a f i e and H i f , 1978 ; Lui et a l . , 1981). b) T e s t o s t e r o n e The dose of t e s t o s t e r o n e enanthate used in t h i s study had p r e v i o u s l y been shown to be ab le to r e s t o r e c i r c u l a t i n g l e v e l s of t e s t o s t e r o n e and normal ize AHH a c t i v i t y in the a d u l t c a s t r a t e male (Sunahara , 1984) as wel l as r e s t o r i n g the l e v e l s of the HCLA b i n d i n g p r o t e i n i n the gonadectomized 5 4 a d u l t male ( F i n l a y s o n , 1983) . There was, however, no ev idence of r e s t o r a t i o n of the HCLA b i n d i n g p r o t e i n l e v e l s with t h i s t reatment in the d i a b e t i c a n i m a l . It cou ld be tha t a l onger t reatment course i s necessary as the r e s t o r a t i o n seen by F i n l a y s o n in the gonadectomized group was a f t e r 10 days ; i t may have occured sooner but a time course was not done. However, i t shou ld be noted tha t the t e s t o s t e r o n e t rea tment was i n i t i a t e d at the same t ime as STZ. c) 3 , 3 , 5 - t r i i o d o t h y r o n i n e The dose of t r i i o d o t h y r o n i n e used in t h i s study has p r e v i o u s l y been shown to r e s t o r e normal t h y r o i d s t a t u s in the d i a b e t i c animal but not cause any o ther a l t e r a t i o n s in the d i a b e t i c s t a t e ( T a h i l i a n i , 1983). There was no e f f e c t of t h i s hormone t reatment on the l e v e l s of the HCLA b i n d i n g p r o t e i n , i n d i c a t i n g that t h y r o i d hormones are not p l a y i n g a r o l e in the r e g u l a t i o n of the HCLA b i n d i n g p r o t e i n , though they do work to c o r r e c t some of the a b n o r m a l i t i e s in p i t u i t a r y growth hormone content seen in the d i a b e t i c animal ( O r t i z - C a r o et a l . , 1984). d) Growth hormone Growth hormone s e c r e t i o n p a t t e r n s show a marked d i f f e r e n c e between the male and female a n i m a l . In the female t h e r e i s a con t inuous r e l e a s e of growth hormone which l eads to s t a b l e , c o n s t a n t l y d e t e c t a b l e l e v e l s in the c i r c u l a t i o n . In the male though , the s e c r e t i o n f o l l o w s an u l t r a d i e n e rhythm which leads to high l e v e l s (peaks) which are r a p i d l y c l e a r e d from the c i r c u l a t i o n , l e a v i n g no d e t e c t a b l e growth hormone ( t roughs) u n t i l the next s e c r e t o r y peak. Th i s rhythmic c y c l e occurs approx imate ly every 3.5 hours in the male r a t . The l e v e l of growth hormone seen dur ing a peak in the male 's c y c l e i s about twice that which i s c o n t i n u o u s l y present in the female (Tannenbaum and M a r t i n , 1976). The f i r s t two exper iments c a r r i e d out with growth hormone were done to r e s t o r e a b s o l u t e c i r c u l a t i n g l e v e l s of growth hormone ( v i a minipumps) which are decreased in d i a b e t i c s , and to mimic the normal male r e l e a s e p a t t e r n ( v i a s . c . i n j e c t i o n s seven t imes per day) which i s f emin i zed in the d i a b e t i c male . Ne i the r of these exper iments r e s u l t e d in the e l e v a t i o n of the HCLA b i n d i n g p r o t e i n to c o n t r o l l e v e l s . In r e t r o s p e c t , t h a t t h e r e was no d i f f e r e n c e between the e f f e c t s of the two t rea tment regimens i s not s u p r i s i n g , s i n c e a s . c . i n j e c t i o n probab ly acts in r a t h e r s i m i l a r manner to a depot p r e p a r a t i o n such as the minipump. That i s , there i s a slow constant d i f f u s i o n i n t o the b l o o d , and no marked peaks and v a l l e y s are seen in the c i r c u l a t o r y system. Thus , the two t reatments probab ly r e s u l t in a s i m i l a r dos ing p a t t e r n . That these t reatments f a i l e d to r e s t o r e the decreased l e v e l s or the HCLA b i n d i n g p r o t e i n , does not negate the theory that growth hormone i s the f e m i n i z i n g f a c t o r i n v o l v e d in the changes seen in l i v e r f u n c t i o n r e l a t e d to d r u g / s t e r o i d metabol ism ( W i l s o n , 1969; 1970; Mode et a l . , 1981; V o c k e n t a n z and V i r g o , 1 9 8 5 ) . I t s presence i s a s s o c i a t e d with f e m i n i z a t i o n and i t s absence causes m a s c u l i n i z a t i o n of d r u g / s t e r o i d metabol ism ( V i r g o , 1985). Th is would e x p l a i n the u l t r a d i e n e p a t t e r n seen in the normal male as some growth hormone (the peak) i s necessary f o r normal growth and development , wh i l e the c l e a r a n c e of growth hormone from the c i r c u l a t i o n (the t rough) a l lows m a s c u l i n i z a t i o n of va r ious p h y s i o l o g i c a l parameters to be m a i n t a i n e d . The maintenance of a low l e v e l of growth hormone in the c i r c u l a t i o n i s probably dependent upon c i r c u l a t i n g t e s t o s t e r o n e l e v e l s (Jansson et al . , 1984) which are capab le of i n h i b i t i n g growth hormone r e l e a s e from the p i t u i t a r y (Ha l l et a l . , 1984). S ince t e s t o s t e r o n e l e v e l s are decreased in d i a b e t e s t h i s i n h i b i t o r y and c l e a r i n g e f f e c t i s not s e e n , l e a d i n g to femi ni zat i on . S ince the s . c . i n j e c t i o n of growth hormone d id not d u p l i c a t e the normal s e c r e t o r y p a t t e r n , we dec ided to t r y i n t r a v e n o u s i n j e c t i o n s to o b t a i n the plasma peaks . The e f f e c t i v e n e s s of i n t ravenous i n j e c t i o n s over e i t h e r s . c . i n j e c t i o n s or cont inuous intavenous i n f u s i o n in d u p l i c a t i n g normal growth hormone p a t t e r n s in the male rat has r e c e n t l y been demonstrated (C la rk et a l . , 1985). The i n e f f e c t i v e n e s s of growth hormone to r e s t o r e the HCLA b i n d i n g p r o t e i n l e v e l s was again seen (Tab les V and V I ) . Th is lack of e f f e c t remained when t e s t s o t e r o n e and i n s u l i n were added to the t rea tment regimen to r e s t o r e a more normal 5 7 p h y s i o l o g y , e . g . t e s t o s t e r o n e to o b t a i n a more normal b a s e l i n e of growth hormone a f t e r the i n j e c t i o n . We were i n j e c t i n g four t imes per day , not the p h y s i o l o g i c a l seven of the u l t r a d i e n e p a t t e r n . Th is may have had an e f f e c t on the lack of r e s t o r a t i o n of HCLA b i n d i n g p r o t e i n l e v e l s a l though n o r m a l i z a t i o n of h e p a t i c drug and s t e r o i d metabol ism has been seen with four i n j e c t i o n s per day (Sket t and Young, 1982). These s t u d i e s were marked by a f u r t h e r c o m p l i c a t i o n . Animals which had been cannu la ted and those n o n - d i a b e t i c c o n t r o l s undergoing t a i l ve in i n j e c t i o n s 4 t imes per day showed markedly reduced l e v e l s of the HCLA b i n d i n g p r o t e i n compared to non-manipulated c o n t r o l s (Tab les V and V I , F i g . 6 ) . The l e v e l of decrease in the n o n - d i a b e t i c t a i l ve in i n j e c t e d c o n t r o l s was approx imate ly 50% from the non-manipu lated c o n t r o l s . The l e v e l s of the HCLA b i n d i n g p r o t e i n in the t a i l ve in i n j e c t e d d i a b e t i c animals (Tab le VI) was a f u r t h e r 50% below the reduced c o n t r o l v a l u e . It thus appeared that these m a n i p u l a t i o n s ( i . e . s u r g e r y and c o n s c i o u s t a i l ve in i n j e c t i o n ) caused a decrease which was a d d i t i v e to that of d i a b e t e s . It has been repor ted (Nakashima et a l . , 1975 ) that the s t r e s s of surgery can lower serum t e s t o s t e r o n e l e v e l s f o r s e v e r a l d a y s . The animals undergoing t a i l ve in i n j e c t i o n s f o u r t imes per day appeared to be undergoing s t r e s s probab ly due to pa in and h a n d l i n g ; presumably t h i s would be s i m i l a r to the s t r e s s of surgery and subsequent i n t o l e r a n c e of the 5 8 c a n n u l a . Th is lower ing of t e s t o s t e r o n e cou ld lead to the decreased l e v e l s of the HCLA b i n d i n g p r o t e i n in c o n t r o l s , and the f u r t h e r decrease in the a l r e a d y depressed d i a b e t i c s . Another a l t e r a t i o n of t e s t o s t e r o n e l e v e l s cou ld be vi a an i n t e r f e r e n c e with the p r o l a c t i n and c o r t i c o t r o p i n i n t e r a c t i o n which leads to adrenal androgen r e l e a s e (Higuch i et a l . , 1984). S ince p r o l a c t i n l e v e l s are decreased in d i a b e t e s , a r e d u c t i o n in the a b i l i t y of t h i s pathway to produce t e s t o s t e r o n e would a l s o be seen . Catecho lamines are e l e v a t e d by s t r e s s and there i s ev idence that an a - a d r e n e r g i c mechanism is i n v o l v e d in the r e g u l a t i o n of growth hormone s e c r e t i o n ( T e r r y and M a r t i n , 1981) ( i . e . ca techo lamines act to cause r e l e a s e ) . However, r a t s reac t to s t r e s s by i n h i b i t i n g the s e c r e t i o n of growth hormone ( T e r r y et al . , 1 977 ) which would s u g g e s t t h a t t h i s f a c t o r i s not of major involvement in the e f f e c t seen . 4) Other s p e c i e s No d e t e c t a b l e b i n d i n g was observed in the o ther s p e c i e s examined f o r the presence of the h e p a t i c HCLA b i n d i n g p r o t e i n . These p a r t i c u l a r s p e c i e s (C57BL/6 mice , DBA/2 mice , guinea p i g s ) were s e l e c t e d due to the i n d i v i d u a l v a r i a t i o n seen amongst the ra ts used in the e x p e r i m e n t s . S i n c e g e n e t i c s are not t h a t we l l worked out i n r a t s , we used mice where the g e n e t i c s are c l e a r l y d e f i n e d , to see i f the re might be an i n d i v i d u a l l ocus with which the HCLA b i n d i n g p r o t e i n s e g r e g a t e d . We chose these s p e c i e s because of the known d i f f e r e n c e s in the Ah locus which c o n t r o l s the s o - c a l l e d T .C .D .D r e c e p t o r , s i n c e there was a sugges t i on that there may be an r e l a t i o n s h i p between t h i s r e c e p t o r and the HCLA b i n d i n g p r o t e i n ( F i n l a y s o n , 1983). The C57BL/6 mice and guinea p igs have a high a f f i n i t y T . C . D . D . r e c e p t o r sys tem, wh i l e the DBA/2 mice posess a very low T . C . D . D r e c e p t o r system. The lack of ev idence f o r the HCLA b i n d i n g p r o t e i n in a l l th ree s p e c i e s i n d i c a t e s that there i s not an a s s o c i a t i o n between the T . C . D . D . r e c e p t o r system and the HCLA b i n d i n g p r o t e i n . It a l so i n d i c a t e s that the HCLA b i n d i n g p r o t e i n may be a rat s p e c i f i c p r o t e i n . 6 0 P R O P O S E D F U T U R E E X P E R I M E N T S A s p r e v i o u s l y m e n t i o n e d , a l o n g e r t i m e c o u r s e o f h o r m o n a l t r e a t m e n t s s h o u l d b e u n d e r t a k e n ( s p e c i f i c a l l y w i t h t e s t o s t e r o n e ) s i n c e i t h a s b e e n s h o w n t h a t i t t a k e s t w o w e e k s f o r t h e H C L A e s t r o g e n b i n d i n g p r o t e i n t o a p p e a r i n h y p o p h y s e c t o m i z e d f e m a l e r a t s . I t c o u l d b e t h a t t h e c o n t r o l m e c h a n i s m i n m a l e d i a b e t i c s i s d a m a g e d a n d t a k e s a l o n g e r a m o u n t o f t i m e ( w i t h t h e p r o p e r r e g u l a t o r y h o r m o n e s ) b e f o r e l e v e l s o f t h e p r o t e i n c a n b e r e g e n e r a t e d . A t i m e c o u r s e o f t h e d e p r e s s i o n o f t h e H C L A b i n d i n g p r o t e i n l e v e l s h o u l d b e u n d e r t a k e n t o s e e h o w q u i c k l y t h e d e p r e s s i o n o c c u r s a n d i f i t c a n b e c o r r e l a t e d w i t h a n y o t h e r a l t e r a t i o n s o c c u r r i n g w i t h i n ( o r s l i g h t y b e f o r e ) t h e s a m e t i m e s p a n . T e s t o s t e r o n e r e p l a c e m e n t s h o u l d b e a t t e m p t e d i n c o n s c i o u s t a i l v e i n i n j e c t e d c o n t r o l a n i m a l s t o f u r t h e r d e l i n e a t e i t s r o l e i n t h e H C L A b i n d i n g p r o t e i n r e g u l a t i o n . A d r e n a l e c t o m y s h o u l d b e p e r f o r m e d o n c o n t r o l a n d d i a b e t i c r a t s t o s e e w h a t e f f e c t c a t e c h o l a m i n e s m a y b e h a v i n g i n t h e r e g u l a t i o n i n t h e n o n - s t r e s s e d a n i m a l , a n d t h e s a m e o p e r a t i o n s h o u l d b e p e r f o r m e d o n t h e s t r e s s e d a n i m a l s t o s e e i f t h e r e a r e f u r t h e r a l t e r a t i o n s , a n d m o r e c o m p l e t e l y e x a m i n e t h e r o l e o f s t r e s s o n t h e H C L A e s t r o g e n b i n d i n g p r o t e i n . L i g a n d s p e c i f i c i t y s t u d i e s s h o u l d b e p e r f o r m e d o n t h e d i a b e t i c a n i m a l s t o s e e i f t h e r e a r e a n y f u n d a m e n t a l a l t e r a t i o n s i n t h e H C L A b i n d i n g p r o t e i n s y s t e m o t h e r t h a n reduced c a p a c i t y . Spontaneous d i a b e t i c B.B. ra t s should be s t u d i e d u s i n g the same assay system to see i f the e f f e c t s of t h e i r d i s e a s e s t a t e p a r a l l e l those seen with STZ or i f the re are d i f f e r e n c e s in these two s t a t e s . If the re a r e , these would have to be i n v e s t i g a t e d to see i f they are a r e s u l t of the chemical used or i f they are due to d i f f e r e n c e s in the p a t h o l o g i e s of the d i s e a s e s . Other t i s s u e s should be examined in c o n t r o l and d i a b e t i c animals to see i f the HCLA b i n d i n g p r o t e i n i s e x c l u s i v e to the l i v e r or i f i t i s more d i f f u s e in d i s t r i b u t i o n , which would have i m p l i c a t i o n s as to i t s p o s s i b l e f u n c t i o n . E n u c l e a t i o n exper iments to examine the exact i n t r a c e l l u l a r d i s t r i b u t i o n of the HCLA b i n d i n g p r o t e i n would be of i n t e r e s t in l i g h t of the c o n t r o v e r s y d e v e l o p i n g over the l o c a l i z a t i o n of es t rogen r e c e p t o r s . F i n a l l y , i s o l a t i o n , f u r t h e r c h a r a c t e r i z a t i o n , and amino a c i d sequenc ing of the HCLA b i n d i n g p r o t e i n should be c a r r i e d o u t . SUMMARY STZ- induced d i a b e t e s causes a decrease in the c a p a c i t y of the HCLA es t rogen b i n d i n g p r o t e i n found in male rat l i v e r without a f f e c t i n g the Kd. Hormonal t reatments i n c l u d i n g i n s u l i n , t e s t o s t e r o n e , t r i i o d o t h y r o n i n e , and growth hormone were i n e f f e c t i v e in r e s t o r i n g the reduced l e v e l s when they were a d m i n i s t e r e d in doses and p a t t e r n s which d u p l i c a t e d normal p h y s i o l o g y . Th i s i n a b i l i t y to r e s t o r e the HCLA es t rogen b i n d i n g p r o t e i n l e v e l s i n d i c a t e s that there i s not a d i r e c t i n t e r p l a y between these hormones and the HCLA l e v e l s in the d i a b e t i c male r a t . The lack of e f f e c t of these hormonal t reatments to r e s t o r e the HCLA es t rogen b i n d i n g p r o t e i n l e v e l s , wh i l e they do r e s t o r e the a l t e r a t i o n s in d r u g / s t e r o i d metabol ism seen in d i a b e t e s , lead us to conc lude that there i s no d i r e c t connec t ion between the HCLA es t rogen b i n d i n g p r o t e i n and the sex-dependent drug metabol ism p a t t e r n s observed in the r a t . A l s o , the HCLA es t rogen b i n d i n g p r o t e i n was not d e t e c t a b l e in the l i v e r c y t o s o l of s e v e r a l o ther s p e c i e s examined i n d i c a t i n g that i t may be rat s p e c i f i c . BIBLIOGRAPHY A n d e r s o n , D.C. C l i n . Endocr i n o l . 3 : 69 , 1974 Appel , M .C . , R o s s i n i , A . A . , W i l l i a m s , R .M. , L i k e , A . A . D i a b e t o l o g i a 15: 327, 1978 A r i s o n , R . N . , C i a c c i o , E . I . , G l i t z e r , M .S . , C a s s a r o , J . 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S t e r o i d B i o c h e m . 16 : 4 3 7 , 1982 APPENDIX I 1) S a t u r a t i o n c u r v e s The d a t a c o n t a i n e d i n t h i s s e c t i o n we re s a m p l e s o f s a t u r a t i o n c u r v e s w h i c h we re u s ed t o g e n e r a t e t h e S c a t c h a r d p l o t s i n t h e R e s u l t s s e c t i o n . The s a t u r a t i o n c u r v e s had t o i n d i c a t e t h a t s p e c i f i c , s a t u r a b l e b i n d i n g o v e r t h e l i g a n d c o n c e n t r a t i o n r a n g e u sed was t a k i n g p l a c e b e f o r e a S c a t c h a r d p l o t c o u l d be g e n e r a t e d w i t h c o n f i d e n c e . F i g u r e A l 3 S p e c i f i c a l l y bound [ H ] - e s t r a d i o l v_s 1 i g a n d 3 c o n c e n t r a t i o n ( [ H ] - e s t r a d i o l ) i n t a i l v e i n i n j e c t e d c i t r a t e c o n t r o l a n i m a l . I n c u b a t i o n s c a r r i e d ou t as d e s c r i b e d i n t h e Me t hod s s e c t i o n . (o o) S p e c i f i c b o u n d , (3————d) n o n - s p e c i f i c b o u n d . G r aph shown i s o f a s i n g l e e x p e r i m e n t and r e p r e s e n t a t i v e o f t h e r e s u l t s f o u n d . 7 1 lO-i 0 5 0 I O O 1 5 0 2 0 0 FREE LIGAND Cnmol /1) F i g u r e A 2 S p e c i f i c a l l y bound [ H ] - e s t r ad i ol v_s l i g a n d c o n c e n t r a t i o n ([ H ] - e s t r a d i o l ) in 4 day STZ- induced d i a b e t i c a n i m a l . Incubat ions were c a r r i e d out as d e s c r i b e d in the Methods s e c t i o n . (o o) S p e c i f i c bound, fci •) n o n - s p e c i f i c bound. Graph shown i s of a s i n g l e experiment and r e p r e s e n t a t i v e of the r e s u l t s f o u n d . 

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