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The high affinity-low capacity androgen binding protein in the hepatic cystol of streptozotocin diabetic… Rodway, Marie R. 1986

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THE HIGH AFFINITY-LOW CAPACITY ANDROGEN BINDING PROTEIN IN THE HEPATIC CYTOSOL OF STREPTOZOTOCIN DIABETIC WISTAR RATS By Marie R. Rodway B . S c . U n i v e r s i t y of A l b e r t a , 1981 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN THE FACULTY OF GRADUATE STUDIES FACULTY OF PHARMACEUTICAL SCIENCES UNIVERSITY OF BRITISH COLUMBIA We accept t h i s t h e s i s as conforming to the require.d s tandard THE UNIVERSITY OF BRITISH COLUMBIA F e b r u a r y , 1986 © Marie R. Rodway, 1986 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 1-6 (3/81) 1 1 ABSTRACT R e c e n t l y s e v e r a l g r o u p s have i d e n t i f i e d and p a r t i a l l y c h a r a c t e r i z e d a h i g h a f f i n i t y - l o w c a p a c i t y a n d r o g e n b i n d i n g p r o t e i n (HALC ABP) i n t he h e p a t i c c y t o s o l o f r a t s , r a b b i t s and h u m a n s . In o r d e r t o f u r t h e r c h a r a c t e r i z e and t o i d e n t i f y t h e p h y s i o l o g i c a l c o n t r o l m e c h a n i s m s o f t h i s p r o t e i n we d i d a s e r i e s o f s t u d i e s i n W i s t a r r a t s . The s y n t h e t i c a n d r o g e n m e t h y l t r i e n o l o n e ( R1881 ) was u s e d as t he l i g a n d i n b i n d i n g s t u d i e s . P r e c i p i t a t i o n w i t h 50% ammonium s u l f a t e r e s u l t e d i n a 2 t o 3 f o l d p u r i f i c a t i o n , bu t d i d n o t e l i m i n a t e t he g l u c o c o r t i c o i d r e c e p t o r t o w h i c h R1881 a l s o b i n d s . F r o z e n s t o r a g e , a t -80°C has no a p p a r e n t e f f e c t on t h e h e p a t i c HALC A B P . S t u d i e s on s e x and age d i f f e r e n c e s showed t h a t R1881 b i n d i n g was p r e s e n t i n ma t u r e f e m a l e s and imma tu r e r a t s a t l e s s t h a n o n e - h a l f t he c o n c e n t r a t i o n f o u n d i n m a t u r e m a l e s . N e o n a t a l c a s t r a t i o n o f m a l e s a p p e a r s t o d e c r e a s e o r e l i m i n a t e t he HALC ABP i n m a t u r e r a t s . No e v i d e n c e o f b i n d i n g o f R1881 t o a n d r o g e n b i n d i n g p r o t e i n s i n b l o o d was seen . The HALC ABP b i n d i n g c a p a c i t y was s i g n i f i c a n t l y r e d u c e d f r o m c o n t r o l l e v e l s i n 4 and 10 day s t r e p t o z o t o c i n ( S T Z , 60 m g / k g , i n t r a v e n o u s ) d i a b e t i c male W i s t a r r a t s ; t h e r e a r e no a p p a r e n t c h a n g e s i n Kd o r i n s t e r o i d s p e c i f i c i t y . i i l A f t e r i n d u c t i o n of d i a b e t e s with STZ, serum i n s u l i n l e v e l s are s i g n i f i c a n t l y decreased in 24 hours-T e s t o s t e r o n e serum l e v e l s and t r i i o d o t h y r o n i n e serum l e v e l s are s i g n i f i c a n t l y decreased 3 days a f t e r STZ i n j e c t i o n . Dampening of the normal peaks of growth hormone s e c r e t i o n in male r a t s i s e v i d e n t 18 hours a f t e r STZ i n j e c t i o n , and c o n t i n u e s p r o g r e s s i v e l y . These changes cor respond to the decrease in b ind ing c a p a c i t y of the h e p a t i c HALC ABP. I f the HALC ABP were r e g u l a t e d by one of these hormones, r e s t o r a t i o n of t h e i r serum l e v e l s should have r e s t o r e d the b i n d i n g c a p a c i t y of the HALC ABP. P a r t i a l r e s t o r a t i o n of the b ind ing c a p a c i t y of the p r o s t a t i c androgen r e c e p t o r was p o s s i b l e with t e s t o s t e r o n e or i n s u l i n in STZ d i a b e t i c r a t s . Attempted r e s t o r a t i o n to c o n t r o l l e v e l s of the h e p a t i c HALC ABP by t reatment of 4 day STZ d i a b e t i c s with the f o l l o w i n g hormones was u n s u c c e s s f u l : i n s u l i n (protamine z i n c i n s u l i n , 10 U/kg s . c . d a i l y , or Toronto i n s u l i n 15 U/kg s . c . twice d a i l y ) ; t e s t o s t e r o n e enanthate (1 mg/kg s . c . d a i l y ) ; t r i i o d o t h y r o n i n e (30 ng/kg s . c . d a i l y ) ; or ovine growth hormone (by minipump 0.02 U/hr f o r 4 days , s . c . 30 ng/dose f o r 7 d a i l y doses , vena cava c a t h e t e r 30 rig/dose f o r 7 d a i l y d o s e s , and t a i l ve in i n j e c t i o n 30 ug/dose, f o r 4 d a i l y dose s ) . I t i s conc luded tha t the HALC ABP i s not r e g u l a t e d by i n s u l i n l e v e l s , by t e s t o s t e r o n e l e v e l s , by T^ l e v e l s , or by GH l e v e l s or the GH male s e c r e t o r y p a t t e r n . S t r e s s 1 V a p p e a r s t o c a u s e a d e c r e a s e i n t h e b i n d i n g c a p a c i t y o f t h e HALC ABP i n a d u l t ma le W i s t a r r a t s . G a i l D. B e l l w a r d , P h . D . Supe r v i s o r V TABLE OF CONTENTS P A G E ABSTRACT i i TABLE OF CONTENTS v LIST OF TABLES x i LIST OF FIGURES x i v LIST OF ABBREVIATIONS xv ACKNOWLEDGEMENTS x v i i INTRODUCTION I . T i s s u e s in which androgen r e c e p t o r s have been s t u d i e d 1 I I . Androgen b i n d i n g p r o t e i n s in l i v e r 4 I I I . Androgen r e c e p t o r s in d i a b e t i c r a t s 7 IV. Changes in hormone l e v e l s and s e c r e t i o n in d i a b e t i c r a t s 1. I n s u l i n 7 2. T e s t o s t e r o n e 8 3. T r i i o d o t h y r o n i n e and t h y r o x i n e 13 4. Growth hormone 14 V. O b j e c t i v e s 14 METHODS AND MATERIALS I . Chemica ls 18 I I . Care and surgery of animals 19 I I I . Treatment of animals 20 V 1 IV. P r e p a r a t i o n of c y t o s o l f o r b i n d i n g assays 1. P r e p a r a t i o n of whole c y t o s o l 22 2. F r a c t i o n a t i o n of whole c y t o s o l 23 3. F r e e z i n g of ammonium s u l f a t e p e l l e t 23 4. Determinat ion of p r o t e i n c o n c e n t r a t i o n 23 V. R a d i o l i g a n d exchange b i n d i n g s t u d i e s 1. Incubat ion of c y t o s o l 24 2. S e p a r a t i o n of bound and f r e e s t e r o i d f r a c t i o n s 25 V I . A n a l y s i s of r a d i o a c t i v i t y 25 V I I . S t a t i s t i c s 26 RESULTS I . The e f f e c t s of ammonium s u l f a t e f r a c t i o n a t i o n of c y t o s o l on the h e p a t i c high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n 1. The e f f e c t of ammonium s u l f a t e f r a c t i o n a t i o n on the Kd and b i n d i n g c a p a c i t y va lues of the high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n 27 2. The presence of the g l u c o c o r t i c o i d r e c e p t o r in the 50 % ammonium s u l f a t e f r a c t i o n a t e d h e p a t i c c y t o s o l of the a d u l t male r a t 29 3. The e f f e c t of 50 % ammonium s u l f a t e f r a c t i o n a t i o n on the optimum i n c u b a t i o n time f o r the high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n assay 33 v i i I I . The e f f e c t of the p e r f u s a t e volume on the r e s u l t s of the high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n assay 33 I I I . The e f f e c t of f r e e z i n g on the h igh a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n 38 IV. Age and sex d i f f e r e n c e s in the presence of the h e p a t i c c y t o s o l i c high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n in r a t s 1. In mature female r a t s 41 2. In immature male and female r a t s 43 3. In c a s t r a t e d male r a t s 43 V. Presence of the high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n in the h e p a t i c c y t o s o l of g e n e t i c a l l y d i a b e t i c B i o B r e e d i n g s t r a i n r a t s . . . . 46 V I . E f f e c t s of s t r e p t o z o t o c i n induced d i a b e t e s on the Kd v a l u e s , b i n d i n g c a p a c i t y , temperature s t a b i l i t y , and s t e r o i d s p e c i f i c i t y of the high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n 1. High a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n b i n d i n g s t u d i e s in c o n t r o l , 4 and 10 day s t r e p t o z o t o c i n d i a b e t i c r a t s 48 2. Temperature s t a b i l i t y of the high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n 58 v i i i 3. E f f e c t of the i n d u c t i o n of s t r e p t o z o t o c i n d i a b e t e s on the s p e c i f i c i t y of the h igh a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n 58 V I I . E f f e c t of hormone replacement on the h e p a t i c c y t o s o l i c high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n 1. E f f e c t s of once d a i l y a d m i n i s t r a t i o n of protamine z i n c i n s u l i n and twice d a i l y a d m i n i s t r a t i o n of Toronto i n s u l i n 59 2. E f f e c t s of t e s t o s t e r o n e enanthate t reatment or t e s t o s t e r o n e enanthate wi th protamine z i n c i n s u l i n t reatment 66 3. E f f e c t s of t r i i o d o t h y r o n i n e t reatment 66 4. E f f e c t s of growth hormone rep lacement a . E f f e c t s of con t inous i n f u s i o n of ov ine growth hormone by osmotic minipump 69 b. E f f e c t s of m u l t i p l e subcutaneous ov ine growth hormone doses 69 c . E f f e c t s of m u l t i p l e doses of ov ine growth hormone us ing a vena cava c a t h e t e r 72 d . E f f e c t s of e ther versus ha lothane general a n a e s t h e s i a in c o n t r o l r a t s 74 e. E f f e c t s of m u l t i p l e t a i l vein i n j e c t i o n s of ov ine growth hormone 74 i x f . E f f e c t s of m u l t i p l e t a i l ve in i n j e c t i o n s of ov ine growth hormone, with d a i l y i n j e c t i o n s of t e s t o s t e r o n e enantha te , s u b c u t a n e o u s l y , and d a i l y i n j e c t i o n s of protamine z i n c i n s u l i n , subcutaneous ly 77 DISCUSSION I . Ammonium s u l f a t e f r a c t i o n a t i o n 79 I I . I n c l u s i o n of t r i a m c i n o l o n e a c e t o n i d e in the h e p a t i c p a r t i a l l y p u r i f i e d c y t o s o l i c high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n assay 81 I I I . A r t i f a c t u a l r e s u l t s from blood c o n t a m i n a t i o n . . . . 82 IV. Kd and b i n d i n g c a p a c i t y va lues of the h e p a t i c high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n 83 V. S p e c i f i c i t y of the h e p a t i c high a f f i n i t y -low c a p a c i t y androgen b i n d i n g p r o t e i n 80 V I . Sex and age d i f f e r e n c e s in the h e p a t i c high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n . . 87 V I I . E f f e c t s of d i a b e t e s on the h e p a t i c and p r o s t a t i c high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t i e n 90 V I I I . N o n - s p e c i f i c e f f e c t s of s t r e p t o z o t o c i n 92 IX. Use of ov ine growth hormone to r e s t o r e h e p a t i c high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n b i n d i n g c a p a c i t y 94 X X. E f f e c t of s t r e s s on the h e p a t i c h igh a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n . . 94 X I . S u b c e l l u l a r l o c a t i o n of the high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n 95 X I I . P o s s i b l e f u n c t i o n of the h e p a t i c high a f f i n i t y -low c a p a c i t y androgen b i n d i n g p r o t e i n 97 FUTURE EXPERIMENTS 98 SUMMARY 103 BIBLIOGRAPHY 106 x i LIST OF TABLES I . The e f f e c t of ammonium s u l f a t e f r a c t i o n a t i o n of whole h e p a t i c c y t o s o l on the high a f f i n i t y -low c a p a c i t y androgen b i n d i n g p r o t e i n a s s a y . . . . 28 I I . The e f f e c t of i n c u b a t i o n t imes from 1.5 to 30 hours on the Kd and b i n d i n g c a p a c i t y va lues of the h e p a t i c c y t o s o l i c high a f f i n i t y -low c a p a c i t y androgen b i n d i n g p r o t e i n 34 I I I . The e f f e c t of d i f f e r e n t l i v e r p e r f u s i o n volumes used p r i o r to high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n assays done in whole and 50 % ammonium s u l f a t e f r a c t i o n a t e d h e p a t i c c y t o s o l . . 37 IV. The e f f e c t of i n c r e a s i n g amounts of t r i a m c i n o l o n e ace ton ide in assays of male and female r a t h e p a t i c c y t o s o l i c h igh a f f i n i t y -low c a p a c i t y androgen b i n d i n g p r o t e i n 42 V. The h e p a t i c high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n content of mature and immature male and female r a t s 44 V I . The e f f e c t of c a s t r a t i o n on the presence of the h e p a t i c c y t o s o l i c high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n in male r a t s 45 V I I . B i n d i n g parameters in B i o B r e e d i n g s t r a i n d i a b e t i c and B i o B r e e d i n g s t r a i n n o n - d i a b e t i c male r a t s compared to c o n t r o l a d u l t male r a t s 47 V I I I . The e f f e c t of 4 and 10 day s t r e p t o z o t o c i n d i a b e t e s x i i on the b i n d i n g parameters of the h e p a t i c c y t o s o l i c h igh a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n 55 IX. ( l , 2 ) T h e e f f e c t of 4 and 10 day s t r e p t o z o t o c i n d i a b e t e s on the temperature s t a b i l i t y of the h e p a t i c c y t o s o l i c high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t i e n of a d u l t male r a t s 56 X. The e f f e c t of 10 day s t r e p t o z o t o c i n d i a b e t e s on the h e p a t i c c y t o s o l i c high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n of male r a t s . . . 60 X I . The e f f e c t of t reatment with r e g u l a r (Toronto) and protamine z i n c i n s u l i n on the h e p a t i c c y t o s o l i c high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n of 4 day s t r e p t o z o t o e i n d i a b e t i c male r a t s 65 X I I . The e f f e c t of t reatment of 4 day s t r e p t o z o t o c i n d i a b e t i c male r a t s with t e s t o s t e r o n e enantha te , or t e s t o s t e r o n e enanthate with protamine z i n c i n s u l i n on the h e p a t i c c y t o s o l i c high a f f i n i t y -low c a p a c i t y androgen b i n d i n g p r o t e i n 67 X I I I . The e f f e c t of t reatment with t r i i o d o t h y r o n i n e on the h e p a t i c c y t o s o l i c high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n of 4 day s t r e p t o z o t o c i n d i a b e t i c male r a t s 68 XIV. The e f f e c t of i n f u s i o n of ov ine growth hormone by minipump on the h e p a t i c c y t o s o l i c high a f f i n i t y -low c a p a c i t y androgen b i n d i n g p r o t e i n of x i i i s t r e p t o z o t o c i n d i a b e t i c male r a t s 70 XV. The e f f e c t of m u l t i p l e subcutaneous doses of ov ine growth hormone on the h e p a t i c c y t o s o l i c high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n of 4 day s t r e p t o z o t o c i n d i a b e t i c male r a t s 71 XVI. The e f f e c t of m u l t i p l e i n t r a v e n o u s doses of ov ine growth hormone by vena cava c a t h e t e r on the h e p a t i c c y t o s o l i c high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n of s t r e p t o z o t o c i n d i a b e t i c male Wis ta r r a t s 73 X V I I . The e f f e c t of ha lothane versus e ther a n a e s t h e s i a on the h e p a t i c c y t o s o l i c high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n of a d u l t male r a t s 75 X V I I I . The e f f e c t of m u l t i p l e i n t raveneous doses of ov ine growth hormone by the t a i l ve in on the h e p a t i c c y t o s o l i c high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n of 4 day s t r e p t o z o t o c i n d i a b e t i c male r a t s 76 XIX. The e f f e c t of subcutaneous a d m i n i s t r a t i o n of 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 with m u l t i p l e t a i l ve in i n j e c t i o n s of ov ine growth hormone on the h e p a t i c c y t o s o l i c h igh a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n of 4 day s t r e p t o z o t o c i n d i a b e t i c male r a t s 78 x i v LIST OF FIGURES PAGE 1. S a t u r a t i o n curves and Sca t chard p l o t f o r b i n d i n g s t u d i e s of the h e p a t i c c y t o s o l i c h igh a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n in 50 % ammonium s u l f a t e f r a c t i o n a t e d c y t o s o l with and wi thout t r i a m c i n o l o n e a c e t o n i d e 30 2. S c a t c h a r d p l o t of the h e p a t i c c y t o s o l i c high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n from f r o z e n 50 % ammonium s u l f a t e f r a c t i o n a t i o n p e l l e t . . 39 3. S a t u r a t i o n curves f o r b i n d i n g s t u d i e s of h e p a t i c c y t o s o l i c high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n from c o n t r o l , 4 and 10 day s t r e p t o z o t o c i n d i a b e t i c a d u l t male r a t s 49 4. S c a t c h a r d p l o t f o r b i n d i n g s t u d i e s of the h e p a t i c c y t o s o l i c high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n in c o n t r o l , 4 and 10 day s t r e p t o z o t o c i n d i a b e t i c a d u l t male r a t s 53 5. S p e c i f i c i t y s t u d i e s of the high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n from c o n t r o l and 10 day s t r e p t o z o t o c i n d i a b e t i c a d u l t male r a t s 62 ABP andros tened ione ANOVA AR C cm c y p r o t e r o n e a c e t a t e DHT e s t r a d i o l F f mol 9 GH 3 [ H ] HALC ABP I C 5 0 Kd kg L mg x v LIST OF ABBREVIATIONS androgen b i n d i n g p r o t e i n A ^ a n d r o s t e n e - 3 , 1 7 - d i o n e a n a l y s i s of v a r i a n c e androgen r e c e p t o r s c e n t i grade c e n t i meter 6 - c h o l o r - l , 2 - d i h y d r o - ( l p , 2 B ) - 3 ' H -c y c l o p r o p a - ( l , 2 ) p r e g n a - l , 4 , 6 -t r i e n e - 3 , 2 0 - d i o n e 5a or 8 - a n d r o s t - 2 - e n , 1 7 p - o l l , 3 , 5 - e s t r a t r i e n e - 3 , 1 7 6 - d i o l a c i d f r e e femto moles gram growth hormone t r i t i ated high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n c o n c e n t r a t i o n r e q u i r e d to d i s p l a c e 50 % of the s p e c i f i c b i n d i n g e q u i l i b r i u m d i s s o c i a t i o n cons tant ki1ogram 1 i t r e m i l l i g r a m s per m i l l i i i t r e mi bo le rone n mL mmol ng nM oGH PZI p roges te rone R1881 s . c . STZ TEDM t e s t o s t e r o n e ( 7 a , 1 7 a - d i m e t h y l [ 1 7 a -3 methyl H ] 1 9 - n o r t e s t o s t e r o n e mi c ro mi 11 i1i t r e mi 11i moles nano gram nanomolar ovine growth hormone protamine z i n c i n s u l i n .4 A p r e g n e n e - 3 , 2 0 - d i o n e m e t h y l t r i e n o l o n e ; 17B-hydroxy-1 7 m e t h y l e s t r a - 4 , 9 , l l - t r i e n - 3 - o n e subcutaneous ly s t r e p t o z o t o c i n ; 2-deoxy-2-((methy n i t r o s o a m i n e ) c a r b o n y l ) - a m i n o ( - D -g lucopyranose) T r i s base , EDTA, d i t h i o t h r e i t o l , sodium molybdate , g l y c e r o l 4 - a n d r o s t e n - 3 - o n e - 1 7 p - o l t r i a m c i n o l o n e a c e t o n i d e 9a- f 1 uoro-HB,16a ,17 ,21- te t ra-o l - p r e g n a - l , 4 - d i e n e - 3 , 2 0 - d i o n e c y c l i c 16,17 a c e t a l with acetone U u n i t s x v i i ACKNOWLEDGEMENTS I would l i k e to thank my s u p e r v i s o r , Dr . G.D. B e l l w a r d , and my commit tee , Dr . B.D. R o u f o g a l i s , Dr . K.M. MacLeod, and Dr . R . J . Ensom. I would a l s o l i k e to thank Bruce A l l e n f o r l i s t e n i n g to me c o m p l a i n . Most e s p e c i a l l y I would l i k e to thank Dr . Geo f f rey Sunahara , who showed me the value of con s i s t e n c y . 1 INTRODUCTION I . T i s s u e s in which androgen r e c e p t o r s have been s t u d i e d I n i t i a l i n v e s t i g a t i o n of androgen r e c e p t o r s (AR) was in t i s s u e p r e v i o u s l y shown to be androgen dependent . Changes i n s i z e or f u n c t i o n of a t i s s u e a f t e r c a s t r a t i o n and r e v e r s a l of these changes with t e s t o s t e r o n e rep lacement was i n d i c a t i v e of androgen dependency. T i s s u e in which AR have been found a r e : the l u n g , comb and w a t t l e of the cock (Dube e t a l , 1 9 7 5 ) , mouse k i d n e y ( P a j u n e n e t a l , 198 2 ) , h a m s t e r sebaceous glands (Adachi and Kano, 1972), the hypothalamus of male m ice , hamsters , guinea p i g s , r a b b i t s , and c a t s (Kato , 1975), r a t s k i n (Eppenderberg and H s i a , 1972), r a t l a c r i m a l g land (Ota et a l , 1985) r a t p r o s t a t e ( K r e i g and V o i g t , 1976 and T r a i s h et al , 1981), r a t S e r t o l i c e l l s (Sanborn et a l , 1984), r a t v e n t r a l and dorsa l p r o s t a t e , ep id idymus , t e s t i s , seminal v e s i c l e and an androgen dependent tumor of the p r o s t a t e (Wi lson and F r e n c h , 1979), r a t b u l b o c a v e r n o s u s , l e v a t o r ani and s k e l e t a l muscle ( K r e i g , 1975, and K r e i g and V o i g t , 1976), r a t hear t ( K r e i g et al , 1978) , r a t hypothalamus ( K a t o , 1975), the a n t e r i o r p i t u i t a r y of mature and immature male r a t s ( K a t o , 1975, and T h i e u l a n t , 1975), the u terus of immature r a t s (G iannopou lous , 1973), female rhesus monkey hear t (McG i l l e t a l , 1980), male and female baboon hear t ( M c G i l l and S h e r i d a n , 1981), in human 2 sexual and non-sexual s k i n , s t r i a t e d muscle and k idney (Des lypere et a l , 1980), human s k i n f i b r o b l a s t s (Keenan et al , 1975 ), human myometrial t i s s u e and mammary cancer t i s s u e (Poortman et a l , 1975, Bryan et a l , 1984), human p r o s t a t e ( M a i n w a r i n g and M i l r o y e t a 1 1973 , D e s l y p e r e e_t al , 1980 T r a i s h et a l , 1981), human benign p r o s t a t i c hypert rophy (Rosen et al , 1 9 7 5 ) , and p r o s t a t i c cancer (Snochowski et a l , 1977, Hawkins et a l , 1981, and K i r d a n i et a l , 1985). From the above l i s t i n g , i t i s e v i d e n t tha t the t i s s u e s which have a t t r a c t e d most i n t e r e s t are c l a s s i c a l sex hormone t a r g e t t i s s u e s . Severa l groups have s t u d i e d AR i s o l a t e d from d i f f e r e n t t i s s u e i n 1 animal to determine t i s s u e s p e c i f i c i t y . Mainwaring and I r v i n g (1973) found t h a t AR in the p r o s t a t e , seminal v e s i c l e s and epididymus of male Sprague-Dawley r a t s had i d e n t i c a l s t e r o i d s p e c i f i c i t y , i d e n t i c a l i s o e l e c t i c p o i n t s ( 5 . 8 ) , and a l l had a sed imenta t ion c o e f f i c i e n t of 8 S. The AR of p r o s t a t e , seminal v e s i c l e s and epididymus of guinea p igs had the same c h a r a c t e r i s t i c s . K r e i g and V o i g t (1976) s t u d i e d the c y t o s o l i c AR of the a d u l t male W i s t a r r a t p r o s t a t e , bu lbocavernosus/1eva to r ani and s k e l e t a l muscle and found Kd's of 1.5, 0 . 7 , and 2.4 nM, r e s p e c t i v e l y , us ing 3 [ H ] 5 a - d i h y d r o t e s t o s t e r o n e (DHT). They found tha t a l l 3 th ree t i s s u e s c o n t a i n e d AR from which [ H]5a-DHT was d i s p l a c e d by 50 to 100 f o l d molar excess of 5a-DHT, t e s t o s t e r o n e and 1 9 - n o r t e s t o s t e r o n e , and by 1000 f o l d molar 3 excess of cypro te rone a c e t a t e and e s t r a d i o l ; but was not d i s p l a c e d by 1000 f o l d molar excess of C o r t i s o l . Wi lson and French (1976) produced e v i d e n c e t h a t AR in d i f f e r e n t t i s s u e s of the r a t were the same. The AR of Sprague-Dawley and Osborn-Mendel r a t t e s t i s , ep id idymus , and p r o s t a t e were i d e n t i c a l . A l though not e x p l i c i t l y s t a t e d i t appears tha t AR in both s t r a i n s of r a t s were a l s o i d e n t i c a l . They found Kd va lues of 0.2 to 0.5 nM us ing 5a-DHT and t e s t o s t e r o n e ; a h igher a f f i n i t y f o r 5a-DHT than f o r t e s t o s t e r o n e ; optimum r e c e p t o r b i n d i n g at pH = 8; changes in the sed imenta t i on c o e f f i c i e n t occured when us ing ammonium s u l f a t e , but no changes in the b i n d i n g p r o p e r t i e s of the r e c e p t o r s ; and s i m i l a r ra tes of a s s o c i a t i o n and d i s s o c i a t i o n of [3H]5a-DHT and [ 3 H ] t e s t o s t e r o n e f o r AR of a l l three t i s s u e s . In these s t u d i e s no t i s s u e s p e c i f i c i t y has been seen . I f no d i f f e r e n c e in the AR e x i s t s , there must be another e x p l a n a t i o n f o r d i f f e r e n c e in t i s s u e s e n s i t i v i t y to d i f f e r e n t androgen ic s t e r o i d s . I t has been found t h a t t i s s u e s e n s i t i v i t y to na tu ra l and s y n t h e t i c androgens depends on the presence and abundance of the AR, the chemical s t r u c t u r e of the androgen a d m i n i s t e r e d , and the presence and abundance of s p e c i f i c androgen m e t a b o l i z i n g enzymes. The chemical s t r u c t u r e of the androgen w i l l determine which enzymes can metabo l i ze i t . Enzymes such as 5 a-reductase and 1 7 6-hydroxysteroid dehydrogenase (which 4 m e t a b o l i z e the androgen) determine s t r u c t u r e , and t h e r e f o r e a f f i n i t y , of the product ( K r e i g and V o i g t , 1976, Des lypere et al , 1980, and Bergink et a l , 1985) . I I . Androgen b i n d i n g p r o t e i n s in l i v e r Androgen b i n d i n g p r o t e i n s (ABP) which have p r o p e r t i e s of r e c e p t o r p r o t e i n s ( s a t u r a b l e , low c a p a c i t y and h igh a f f i n i t y b i n d i n g of androgens) have been s t u d i e d in the l i v e r of r a b b i t s , r a t s and humans. Because p h y s i o l o g i c a l f u n c t i o n has not been determined , we w i l l r e f e r to t h i s androgen b i n d i n g p r o t e i n as "HALC ABP", u n l e s s the author of the r e f e r e n c e d paper has i d e n t i f i e d i t as the h e p a t i c AR. The h e p a t i c c y t o s o l i c AR of female New Zea land whi te r a b b i t s has been s t u d i e d by E i s e n f e l d et al , and Sheets et a l , 1985. They found a Kd of 0 . 3 - 0 . 9 nM and a b i n d i n g c a p a c i t y of 79 fmol/mg. Methyl t r i en ol on e (R1881), 5<x-DHT, t e s t o s t e r o n e , and e s t r a d i o l , in descending order of a f f i n i t y , bound to the AR. P roges terone and d i e t h y l s t i 1 b e s t e r o l d id not b i n d . Th i s p r o t e i n was p r e s e n t in immature females and in a d u l t males in c o n c e n t r a t i o n s about o n e - h a l f tha t seen in mature female r a b b i t s . Human h e p a t i c c y t o s o l i c AR have been s t u d i e d by Aten and E i s e n f e l d , 1983, and by B a n n i s t e r et a l , 1985b. Aten and E i s e n f e l d repor ted a Kd of 1-2 nM, and a b i n d i n g c a p a c i t y of 30-50 fmol/mg p r o t e i n . The sex of the p a t i e n t s 5 from which the b i o p s i e s were taken was not r e p o r t e d . B a n n i s t e r et a l , 1985b, r e p o r t e d r e s u l t s of n u c l e o s o l i c and c y t o p l a s m i c AR b i n d i n g s t u d i e s . The c y t o s o l i c AR had a Kd va lue of 1.42 + 0.30 nM and a b i n d i n g c a p a c i t y of 103 _+ 31 fmol/mg p r o t e i n . The order of a f f i n i t y f o r s t e r o i d s was: mibo lerone > 5a-DHT > c y p r o t e r o n e a c e t a t e > t e s t o s t e r o n e > progesterone > e s t r a d i o l . A t o t a l of 8 p a t i e n t s were b i o p s i e d , 2 were f e m a l e s . Reports c o n c e r n i n g ABP in r a t l i v e r c y t o s o l appeared f i r s t i n the e a r l y 1970 ' s ( M i l i n and Roy, 1973, Roy e t a l , 1974, and G u s t a f s s o n , 1975). The i n t e r p r e t a t i o n of these s t u d i e s was c o m p l i c a t e d by the metabol ism of l i g a n d s , t r a n s f o r m a t i o n of the ABP, and the b i n d i n g of l i g a n d s to an es t rogen-androgen b i n d i n g p r o t e i n p resent in the l i v e r of male r a t s (Gusta fsson et al , 1975, Kyakumoto et a l , 1984, Lev inson and Decker , 1985, and Sunahara et a l , 1985). These c o m p l i c a t i o n s l e d to i n a c c u r a t e e s t i m a t i o n s by S c a t c h a r d a n a l y s i s of Kd va lues and b i n d i n g c a p a c i t i e s . In o rder to overcome these d i f f i c u l t i e s , molybdate , R1881, and t r i a m c i n o l o n e ace ton ide were i n c o r p o r a t e d i n t o the ABP a s s a y . Sodium molybdate was e f f e c t i v e in p r e v e n t i n g the t r a n s f o r m a t i o n of the ABP (rev iewed by Dahmer et a l , 1984). One of the most important changes to the h e p a t i c ABP assay was the i n t r o d u c t i o n of s y n t h e t i c androgens used as r a d i o l a b e l e d and competing s t e r o i d s in r a d i o l i g a n d exchange b i n d i n g s t u d i e s . They were m in ima l l y m e t a b o l i z e d and d id 6 not b i n d to the sex s t e r o i d b i n d i n g g l o b u l i n in b l o o d . R1881 was i n t r o d u c e d f i r s t and i s most w ide ly used . I t i s m i n i m a l l y m e t a b o l i z e d in r a t p r o s t a t i c (Bonne and Raynaud, 1975 and 1976) , and h e p a t i c c y t o s o l (Decker and L e v i n s o n , 1983, Lev inson and Decker , 1985, and Turocy et a l , 1985) . The use of R1881 and of mibo lerone in ABP assays r e v e a l e d two b i n d i n g s i t e s , with d i f f e r e n t a f f i n i t i e s f o r R1881 and t h e r e f o r e d i f f e r e n t Kd va lues (Kyakumoto et a l , 1984, Decker and L e v i n s o n , 1985, Sunahara et al , 1985, and B a n n i s t e r et a l , 1 9 8 5 a ) . P r e v i o u s work done u s i n g R1881 in p r o s t a t i c c y t o s o l a l s o showed two b i n d i n g s i t e s . In the p r o s t a t e the second b i n d i n g s i t e was the p r o g e s t i n r e c e p t o r (Zava et a l , 1979). Lev inson and Decker , 1985, demonst ra ted the presence of the g l u c o c o r t i c o i d r e c e p t o r and absence of a p r o g e s t i n r e c e p t o r in r a t h e p a t i c c y t o s o l , so i t i s b e l i e v e d tha t the second b i n d i n g p r o t e i n of R1881 in h e p a t i c c y t o s o l i s the g l u c o c o r t i c o i d r e c e p t o r . T r i a m c i n o l o n e a c e t o n i d e , a s y n t h e t i c g l u c o c o r t i c o i d , and C o r t i s o l have been used in 100-1000 f o l d molar excess c o n c e n t r a t i o n s in order to e l i m i n a t e the b i n d i n g of r a d i o l a b e l e d and c o m p e t i t o r l i g a n d to the p r o g e s t i n r e c e p t o r i n p r o s t a t e c y t o s o l , and the g l u c o c o r t i c o i d r e c e p t o r in h e p a t i c c y t o s o l (Zava e t al , 1979, E i s e n f e l d et a l , 1983, and Aten e t a l , 1 9 8 3 ) . S a t u r a t i o n c u r v e s of s p e c i f i c R1881 b i n d i n g and S c a t c h a r d a n a l y s i s suggest 1 R1881 b i n d i n g s i t e , so c a l c u l a t i o n of the Kd and maximum b i n d i n g c a p a c i t y 7 has been s i m p l i f i e d to l i n e a r r e g r e s s i o n a n a l y s i s of S c a t c h a r d p l o t s . S t u d i e s done to determine the n e c e s s i t y of both t r i a m c i n o l o n e a c t e t o n i d e and C o r t i s o l i n the i n c u b a t i o n of the HALC ABP assay showed tha t 100 f o l d molar excess was adequate to e l i m i n a t e the i n t e r f e r e n c e of the g l u c o c o r t i c o i d r e c e p t o r in the assay of the h e p a t i c c y t o s o l i c HALC ABP in male a d u l t Wis tar r a t s (Sunahara, 1984). As a r e s u l t of the above m o d i f i c a t i o n s to the h e p a t i c ABP a s s a y , s e v e r a l papers have been p u b l i s h e d which d e s c r i b e i n i t i a l c h a r a c t e r i z a t i o n s t u d i e s of the r a t h e p a t i c ABP (Kyakumoto et a l , 1984, Lev inson and Decker , 1985, Sunahara et al , 1985, and B a n n i s t e r et a l , 1985a). I I I . Androgen r e c e p t o r s in d i a b e t i c r a t s In 1980, Tesone et al examined the e f f e c t of s t r e p t o z o t o c i n (STZ, 65 mg/kg), 14 days a f t e r i n d u c t i o n of d i a b e t e s on AR in p r o s t a t i c c y t o s o l . A s i g n i f i c a n t decrease i n number of AR, p a r t i a l l y r e v e r s i b l e wi th t e s t o s t e r o n e (500 u g / r a t subcutaneous ly ( s . c . ) d a i l y ) or protamine z i n c i n s u l i n (PZI , 2 U/rat s . c . d a i l y ) t reatment was seen . A s s o c i a t i o n c o n s t a n t s (Ka) d id not change s i g n i f i c a n t l y . The a s s o c i a t i o n contant f o r the c o n t r o l group was 0.13 + 0.01 nM; f o r the d i a b e t i c group was 0.17 _+ 0.02 nM; f o r the i n s u l i n t r e a t e d d i a b e t i c group was 0.10 + 0.02 nM; and f o r 8 the t e s t o s t e r o n e t r e a t e d d i a b e t i c group was 0.12 + 0.01 nM. B i n d i n g c a p a c i t y va lues were 94.2 + 11.3 fmol/mg p r o t e i n f o r the c o n t r o l group; 12.6 _+ 3.0 fmol/mg p r o t i e n f o r the d i a b e t i c g r o u p , 18 .3 + 2.4 fmol/mg p r o t e i n f o r the i n s u l i n t r e a t e d d i a b e t i c group, and 41.1 +_ 3.1 fmol/mg p r o t e i n f o r the t e s t o s t e r o n e t r e a t e d d i a b e t i c group. Hense, the s e n s i t i v i t y of the p r o s t a t i c AR to t e s t o s t e r o n e , and to a l e s s e r e x t e n t , i n s u l i n , has been demonst ra ted . IV. Changes in hormone l e v e l s and s e c r e t i o n in d i a b e t i c r a t s 1. I n s u l i n Induc t ion of d i a b e t e s with STZ causes a wide range of hormonal changes . The i n i t i a l and most obv ious change i s the change in the amount of a v a i l a b l e i n s u l i n in the p a n c r e a s . STZ induces d i a b e t e s by caus ing degranul a t i o n and n e c r o s i s of 8 p a n c r e a t i c c e l l s . In mature male Wis ta r r a t s made d i a b e t i c with a dose of 65 mg/kg STZ, l a r g e amounts of i n s u l i n were r e l e a s e d i n t o the blood at around seven hours post i n j e c t i o n . Excess i n s u l i n was s t i l l p resent in the b lood at 10 hours post i n j e c t i o n , as the b lood g lucose l e v e l s remained d e p r e s s e d . At 24 hours post i n j e c t i o n p a n c r e a t i c i n s u l i n content was about 5 % of the normal v a l u e , serum immunoreact ive i n s u l i n was at f a s t i n g l e v e l s , 9 and serum g lucose was 300-400 mg/100 mL, c o n t r o l serum g l u c o s e i s under 100 mg/100 mL ( J u n o d , e t a l , 1 9 6 7 ) . Serum i n s u l i n in male 4 day STZ d i a b e t i c r a t s was s i g n i f i c a n t l y reduced from c o n t r o l v a l u e s , 25.9 + 3 . 7 , and 14.1 + 1.6 uUnits/mL, r e s p e c t i v e l y (Warren et al , 1983) . 2. T e s t o s t e r o n e Decreases in serum and t e s t i c u l a r l e v e l s of t e s t o s t e r o n e in STZ d i a b e t i c r a t s have been wel l documented. Howland and Zebrowski (1976) s t u d i e d STZ (70 mg/kg or 65 mg/kg) d i a b e t e s in Sprague-Dawley r a t s . They found a s i g n i f i c a n t r e d u c t i o n in serum c o n c e n t r a t i o n of t e s t o s t e r o n e ( c o n t r o l 4.33 + 0 .89 , and STZ d i a b e t i c 1.08 + 0.10 ng/mL), three weeks a f t e r i n d u c t i o n of d i a b e t e s with STZ. Tesone et a l , 1976, s t u d i e d the e f f e c t s of STZ (50 mg/kg) d i a b e t e s on the serum and t e s t i c u l a r l e v e l s of t e s t o s t e r o n e of a d u l t male W i s t a r r a t s and saw a s i g n i f i c a n t decrease in serum t e s t o s t e r o n e , which cou ld be p a r t i a l l y r e s t o r e d with i n s u l i n t r e a t m e n t , and a s i g n i f i c a n t decrease in t e s t i c u l a r t e s t o s t e r o n e , which cou ld be r e s t o r e d with i n s u l i n t r e a t m e n t . Serum l e v e l s of t e s t o s t e r o n e in c o n t r o l , d i a b e t i c , and d i a b e t i c t r e a t e d with PZI (0 .5 U/rat s . c d a i l y ) were, r e s p e c t i v e l y , 486 + 19, 171 + 9, and 290 + 15 ng/100 mL, measured 15 days a f t e r i n d u c t i o n of d i a b e t e s , or 15 days a f t e r i n i t i a t i o n of i n s u l i n t r e a t m e n t . 1 0 T e s t i c u l a r l e v e l s of t e s t o s t e r o n e f o r c o n t r o l , d i a b e t i c and i n s u l i n t r e a t e d d i a b e t i c were, r e s p e c t i v e l y , 131 + 10, 30 +_ 5, and 111 + 7 ng/g of t e s t i s . Paz e t a l , 1978, s t u d i e d serum and t e s t i c u l a r l e v e l s of t e s t o s t e r o n e in male a l b i n o r a t s made d i a b e t i c with STZ (15 mg/rat) and found s i g n i f i c a n t decreases in serum t e s t o s t e r o n e in d i a b e t i c compared to c o n t r o l r a t s at 6 to 7 weeks a f t e r i n d u c t i o n of d i a b e t e s , which was p a r t i a l l y r e s t o r e d with i n s u l i n t r e a t m e n t . They found serum l e v e l s in c o n t r o l , d i a b e t i c , and d i a b e t i c t r e a t e d wi th 2 IU of PZI s . c . d a i l y , o f , r e s p e c t i v e l y , 2.4 + 0 . 4 , 0.9 + 0 . 3 , and 1.1 _+ 0.2 ng/mL. T e s t i c u l a r l e v e l s of t e s t o s t e r o n e were a l s o s i g n i f i c a n t l y reduced and p a r t i a l l y r e s t o r e d with i n s u l i n t r e a t m e n t . Va lues f o r c o n t r o l , STZ, and STZ t r e a t e d wi th PZI were, r e s p e c t i v e l y , 110 + 16, 38 + 8, and 49 + 4 ng/g of t e s t i s . Baxter et a l , 1981, s t u d i e d the e f f e c t of STZ (100 mg/kg) d i a b e t e s in mature male Wis ta r r a t s . He found a s i g n i f i c a n t r e d u c t i o n in serum l e v e l s of t e s t o s t e r o n e 1 week a f t e r i n d u c t i o n of d i a b e t e s with d i a b e t e s and found t h a t the r e d u c t i o n was i n s u l i n r e v e r s i b l e . Serum l e v e l s of t e s t o s t e r o n e f o r c o n t r o l , d i a b e t i c , and d i a b e t i c t r e a t e d with 10-12 U d a i l y of Monocomponent A c t r a p i d and Lente i n s u l i n a d m i n i s t e r e d tw ice d a i l y , were r e s p e c t i v e l y , 9 .0 +_ 1.3, 3.9 + 0 . 8 , and 7.7 + 1.1 nM. Murray e t al , 1981, s t u d i e d serum t e s t o s t e r o n e l e v e l s 11 in a d u l t male W i s t a r s r a t s made d i a b e t i c w i th 65 mg/kg STZ. They found s i g n i f i c a n t r e d u c t i o n s from c o n t r o l at 4 weeks a f t e r i n j e c t i o n of STZ. Cont ro l l e v e l s were 4.11 + 0.65 ng/mL, l e v e l s at 2, 3, and 4 weeks a f t e r i n j e c t i o n of STZ, w e r e , r e s p e c t i v e l y , of 2.41 + 0 . 6 4 , 3 .49 + 0 . 9 , and 1.41 + 0.58 ng/mL. Rats t r e a t e d wi th 1-2 U/lOOg PZI had l e v e l s of 3.71 + 0.27 ng/mL which was not d i f f e r e n t from c o n t r o l l e v e l s . In 1980 Tesone et al measured serurn t e s t o s t e r o n e l e v e l s in a d u l t male Wis ta r c o n t r o l , d i a b e t i c (65 mg/kg S T Z ) , and i n s u l i n (2 IU PZI s . c d a i l y ) t r e a t e d d i a b e t i c r a t s and found a s i g n i f i c a n t r e d u c t i o n p a r t i a l l y r e v e r s i b l e wi th i n s u l i n t reatment in STZ d i a b e t i c s . Measurements were made 28 days a f t e r i n d u c t i o n of d i a b e t e s . Values were 540 + 64, 238 + 37, and 358 + 18 ng/100 mL, r e s p e c t i v e l y . T e s t i c u l a r t e s t o s t e r o n e l e v e l s were s i g n i f i c a n t l y reduced i n d i a b e t i c s and reversed with i n s u l i n . In the same order va lues were 154 + 13, 41 + 5, and 142 _+ 9 ng/g of t e s t i s . Skeet et al , 1984, showed serum t e s t o s t e r o n e l e v e l s in a d u l t male Wis ta rs of 1.8 + 1.0, and a s i g n i f i c a n t decrease in STZ (60 mg/kg) d i a b e t i c s to 1.0 + 0.03 mg/mL, measured 3 days a f t e r i n d u c t i o n of d i a b e t e s with STZ. Other models of d i a b e t e s a l s o show s i g n i f i c a n t r e d u c t i o n in serum t e s t o s t e r o n e and t e s t i c u l a r l e v e l s of t e s t o s t e r o n e . Howland and Zebrowski (1976) showed a 1 2 s i g n i f i c a n t r e d u c t i o n in serum t e s t o s t e r o n e in a l l o x a n (15 mg/100 g) d i a b e t e s . Va lues in c o n t r o l and d i a b e t i c r a t s were, r e s p e c t i v e l y , 4.33 + 0 .89 , and 1.54 + 0.34 ng/mL. Warren et al , 1983, showed decreased serum t e s t o s t e r o n e l e v e l s in g e n e t i c a l l y d i a b e t i c ( B i o B r e e d i n g ) r a t s as compared to i n t e r n a l c o n t r o l s , or e x t e r n a l , W i s t a r , c o n t r o l s . I n t e r n a l c o n t r o l s to B i o B r e e d i n g r a t s a l s o have serum t e s t o s t e r o n e l e v e l s s i g n i f i c a n t l y reduced from Wis ta r c o n t r o l l e v e l s . Values were 1.16 + 0 .44 , 2.99 + 0 .42 , and 6.4 _+ 1.25 ng/mL, f o r d i a b e t i c B i o B r e e d i n g , n o n - d i a b e t i c B i o B r e e d i n g , and Wis tar c o n t r o l male r a t s , r e s p e c t i v e l y . Serum t e s t o s t e r o n e l e v e l s in B i o B r e e d i n g r a t s were measured 3 days a f t e r s t o p p i n g i n s u l i n t r e a t m e n t . In a l l s t u d i e s , d i a b e t e s , from STZ, a l l o x a n or g e n e t i c a l l y de termined , r e s u l t e d in a s i g n i f i c a n t decrease in serum and t e s t i c u l a r 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 a d m i n i s t e r e d in a dose of 3 to 12 U/day/rat appeared to c o m p l e t e l y reverse the decreased serum t e s t o s t e r o n e l e v e l s ; 2 U d a i l y , however, on ly p a r t i a l l y r e s t o r e d serum t e s t o s t e r o n e l e v e l s . Complete r e s t o r a t i o n of t e s t i c u l a r content of t e s t o s t e r o n e has been r e p o r t e d with as l i t t l e as 0.5 U/rat d a i l y , and with 2 U/rat d a i l y ; however, 2 U/rat/day has a l s o been r e p o r t e d to only p a r t i a l l y r e s t o r e t e s t i c u l a r amounts of t e s t o s t e r o n e . The r e s t o r a t i o n of serum and t e s t i c u l a r amounts of t e s t o s t e r o n e does not appear to be determined by the amount of STZ used to induce d i a b e t e s in the r a t . Decreased b i o s y n t h e s i s of t e s t o s t e r o n e may a l s o occur in c h e m i c a l l y d i a b e t i c r a t s . In I n s t i t u t o de F i s i o l o g i a s t r a i n white r a t s which had 95 % of the pancreas removed, F o g l i a et a l , 1969, have shown decreased 14 t e s t o s t e r o n e b i o s y n t h e s i s . Percentage of C p roges te rone t rans formed to t e s t o s t e r o n e was over 3.5 % in c o n t r o l r a t s and 0 to 3.5 % in r a t s with f a s t i n g b lood g lucose over 120 mg/mL. Decreases in serum and t e s t i c u l a r t e s t o s t e r o n e l e v e l s , p a r t i a l l y r e v e r s i b l e wi th i n s u l i n t rea tment occur in STZ d i a b e t i c male r a t s . S i g n i f i c a n t decreases in t e s t o s t e r o n e s y n t h e s i s occurs in pancrea tec tomized d i a b e t i c male r a t s and may occur in o ther models of d i a b e t e s . 3. T r i i o d o t h y r o n i n e and t h y r o x i n e Jenn ings (1984) induced d i a b e t e s with STZ (65 mg/kg) in mature male Sprague-Dawley r a t s . He showed a decrease i n serum l e v e l s of t h y r o x i n e ( c o n t r o l , 3.7 _+ 0.4 u g / d L , 3 days post STZ i n j e c t i o n - e a r l i e s t time of s i g n i f i c a n t di f f e r e n c e - 2 . 1 _+ 0.4 ng/dL) and t r i i o d o t h y r o n i n e ( c o n t r o l , 35 HH 3 ng/dL, 2 days post STZ i n j e c t i o n - e a r l i e s t time of s i g n i f i c a n t di f f e rence-24 _+ 4 ng/dL) . Using a i s o l a t e d p e r f u s e d r a t l i v e r , he showed a p r o g r e s s i v e decrease i n h e p a t i c t r i i o d o t h y r o n i n e p r o d u c t i o n (from 56 + 11 to 12 +_ 4 ng/hour a f t e r 5 d a y s ) . The r e d u c t i o n i n p r o d u c t i o n was f i r s t s i g n i f i c a n t l y d i f f e r e n t a f t e r 2 days . The 1 4 r e d u c t i o n in t r i i o d o t h y r o n i n e p r o d u c t i o n was p r i m a r i l y due to reduced t h y r o x i n e to t r i i o d o t h y r o n i n e c o n v e r s i o n (3.27 + 0.53 to 0.75 + 0.21 %, on the 5th day of d i a b e t e s ) . D i a b e t i c r a t s t r e a t e d with i n s u l i n (2 .5 07100 g, L e n t e ) , produced serum t h y r o x i n e and t r i i o d o t h y r o n i n e l e v e l s equal to c o n t r o l s . I n s u l i n t reatment a l s o r e s t o r e d c o n v e r s i o n of t h y r o x i n e to t r i i o d o t h y r o n i n e , and t r i i o d o t h y r o n i n e p r o d u c t i o n to c o n t r o l l e v e l s , i n the i s o l a t e d per fused r a t l i v e r . Treatment of d i a b e t i c r a t s w i th t h y r o x i n e (1 ug/100 g d a i l y ) r e s t o r e s serum t h y r o x i n e l e v e l s and t h y r o x i n e to t r i i o d o t h y r o n i n e c o n v e r s i o n , and t h e r e f o r e serum t r i i o d o t h y r o n i n e l e v e l s . P i t t m a n e t a l , 1981, showed a s i g n i f i c a n t d e c r e a s e in serum t h y r o x i n e and t r i i o d o t h y r o n i n e l e v e l s in mature male Sprague-Dawley r a t s made d i a b e t i c w i th 50 mg/kg STZ, but not u n t i l 8 days a f t e r i n d u c t i o n of STZ d i a b e t e s . A s i g n i f i c a n t r e d u c t i o n of t h y r o x i n e to t r i i o d o t h y r o n i n e c o n v e r s i o n in l i v e r homogenates was seen a f t e r 3 days of STZ d i a b e t e s . Mitsuma and Nog imor i , 1982, s t u d i e d the e f f e c t s of STZ (60 mg/kg) d i a b e t e s on the h y p o t h a l a m i c - p i t u i t a r y - t h y r o i d a x i s in a d u l t male d i a b e t i c Wis ta r r a t s . Immunoreactive t h y r o t r o p i n r e l e a s i n g hormone in the hypothalamus was reduced a f t e r 4 weeks of d i a b e t e s . I n s u l i n t reatment (2 U z i n c i n s u l i n i .m . d a i l y ) , p a r t i a l l y r e s t o r e d t h i s . Values f o r c o n t r o l , d i a b e t i c , and i n s u l i n t r e a t e d d i a b e t i c a r e , r e s p e c t i v e l y , 4.4 + 0 . 2 , 3.2 + 0 .2 , 3.7 + 0.3 1 5 ng/hypotha lamus. Basal t h y r o t r o p i n l e v e l s were s i g n i f i c a n t l y reduced a t the f i r s t measurement a f t e r i n d u c t i o n of d i a b e t e s , which was 2 weeks. Amounts were p a r t i a l l y r e s t o r e d wi th i n s u l i n t r e a t m e n t , va lues f o r c o n t r o l , d i a b e t i c , and i n s u l i n t r e a t e d d i a b e t i c a r e , r e s p e c t i v e l y 260 + 22, 160 +_ 14, 196 + 13 ng/mL. Responses of immunoreacti ve t h y r o t r o p i n r e l e a s i n g hormone and plasma t h y r o t r o p i n responses to c o l d were s i g n i f i c a n t l y reduced 2 weeks a f t e r the i n d u c t i o n of d i a b e t e s , and t h i s was p a r t i a l l y r e v e r s e d w i t h i n s u l i n t r e a t m e n t . Plasma t h y r o x i n e , 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 were s i g n i f i c a n t l y decreased ( r e s p e c t i v e l y , 3.6 + 0.3 ng/dL, 3 5 + 4 ng/dL, 2.3 + 0 .4 ng/dL) f rom c o n t r o l amounts ( 5 . 0 _+ 0.4 u g/dL, 53 _+ 5 ng/dL, and 3.4 + 0.6 ng/dL) , at two weeks a f t e r i n d u c t i o n of d i a b e t e s wi th STZ, which was the f i r s t t ime p o i n t i n t h i s s t u d y . I n s u l i n t reatment r e s t o r e d plasma l e v e l s to c o n t r o l v a l u e s ( t h y r o x i n e 3.8 +_ 3 ug/dL, t r i i o d o t h y r o n i n e 48 _+ 4 ng/dL, and 3,3 ' - d i i odothy ron i ne 3.0 _+ 0.6 ng/dL) . The r e d u c t i o n in t r i i o d o t h y r o n i n e and t h y r o x i n e and in t h y r o x i n e to t r i i o d o t h y r o n i n e c o n v e r s i o n c l e a r l y o c c u r s , and i s i n s u l i n r e v e r s i b l e . How soon these decreases become p h y s i o l o g i c a l l y r e l e v a n t a f t e r STZ i n d u c t i o n of d i a b e t e s i s unknown, a l though they are measurable in the per fused r a t l i v e r at 2 days a f t e r STZ i n j e c t i o n . 1 6 4. Growth hormone The p a t t e r n of GH r e l e a s e was changed, and the mean 8 hour plasma l e v e l of GH was decreased i n 15 day STZ (70 mg/kg) male d i a b e t i c W i s t a r r a t s ( O r t i z - C a r o e t a l , 1 9 8 4 ) . Normal mature male r a t s e x h i b i t an u l t r a d i a n p a t t e r n of r e l e a s e (Tannenbaum, 1976) which was dampened by STZ d i a b e t e s so t h a t the p a t t e r n more c l o s e l y resembles the more c o n s t a n t femin ine p a t t e r n of r e l e a s e . T h i s change can be seen 18 hours a f t e r i n j e c t i o n and c o n t i n u e s p r o g r e s s i v e l y . A decrease in p i t u i t a r y GH was a l s o seen . Reduced pu l ses of GH may be mediated by s o m a t o s t a t i n (Tannebaum, 1981). I n s u l i n (15 U/kg twice a day f o r 12 days) r e s t o r e d the normal r e l e a s e p a t t e r n ( O r t i z - C a r o et a l , 1984). The cont inuous presence of t e s t o s t e r o n e i s r e q u i r e d to ma in ta in the low GH b a s e l i n e l e v e l s in a d u l t male r a t s . S t u d i e s done wi th n e o n a t a l l y c a s t r a t e d male and female r a t s suggest an i m p r i n t i n g of the p a t t e r n of GH r e l e a s e by neonatal t e s t o s t e r o n e surges ( J a n s s o n , et al 1984). In male r a t s STZ d i a b e t e s f e m i n i z e s GH r e l e a s e p a t t e r n s . V. O b j e c t i v e s The long s t a n d i n g i n t e r e s t of t h i s l a b o r a t o r y has been in h e p a t i c drug metabo l i sm; in p a r t i c u l a r , i n the sex d i f f e r e n c e s of some h e p a t i c drug and s t e r o i d m e t a b o l i z i n g presence of androgens in the male r a t . Androgens are b e l i e v e d to act by b i n d i n g with a c y t o p l a s m i c r e c e p t o r . If the d i f f e r e n c e in these enzyme a c t i v i t i e s i s due to l o c a l , as opposed to c e n t r a l , a c t i v i t y of androgen, one would expect an AR in the l i v e r . An h e p a t i c androgen b i n d i n g p r o t e i n with the c h a r a c t e r i s t i c s of a r e c e p t o r p r o t e i n has been demonstrated (Kyakumoto et a l , 1984, Lev inson and D e c k e r , 1 9 8 5 , S u n a h a r a e t a l , 1985 , and B a n n i s t e r e t a l 1985). If the sex d i f f e r e n c e depends on the h e p a t i c AR, and t h e r e f o r e l o c a l a c t i o n of androgens, one would expect age and sex dependency of the AR tha t c o r r e l a t e s with age and sex dependency of the sex dependent enzyme a c t i v i t i e s . I t i s wel l known tha t f e m i n i z a t i o n of sex dependent enzyme a c t i v i t i e s occurs in male r a t s which are made d i a b e t i c . Sunahara et a l , 1985, demonstrated a change in the h e p a t i c r e c e p t o r - l i k e b i n d i n g p r o t e i n in the STZ (60 mg/kg) induced d i a b e t i c male W i s t a r r a t . Changes in p a t t e r n of s e c r e t i o n or abso lu te amounts of hormones s e c r e t e d are a l s o known to occur in acute d i a b e t e s ; the h e p a t i c HALC ABP may be r e g u l a t e d by one or more of these hormones. In order to f u r t h e r c h a r a c t e r i z e and i n v e s t i g a t e the r e g u l a t i o n of the HALC ABP, and to determine i f there was a c o r r e l a t i o n between sex dependent enzyme a c t i v i t i e s and the HALC ABP under d i f f e r e n t p h y s i o l o g i c a l c o n d i t i o n s , s t u d i e s us ing 50 % ammonium s u l f a t e f r a c t i o n a t e d c y t o s o l were done t o : 1) re-examine age and sex s p e c i f i c i t y of the 1 8 were done t o : 1) re-examine age and sex s p e c i f i c i t y of the HALC ABP; 2) determine the reason f o r the apparent decrease i n the b i n d i n g c a p a c i t y of the HALC ABP in STZ d i a b e t i c ma les ; 3) and to attempt r e s t o r a t i o n of t h i s decreased b i n d i n g c a p a c i t y of the HALC ABP. S p e c i f i c a l l y , the o b j e c t i v e s of t h i s s tudy were : 1) to study the presence of the h e p a t i c HALC ABP in mature and immature male and female Wis ta r r a t s , i n mature c a s t r a t e d males , in n e o n a t a l l y c a s t r a t e d males , and in n e o n a t a l l y c a s t r a t e d t e s t o s t e r o n e t r e a t e d male r a t s ; 2) to study the e f f e c t of d i a b e t e s induced by STZ on the temperature s t a b i l i t y , b i n d i n g s p e c i f i c i t y , and Kd and b i n d i n g c a p a c i t y v a l u e s , determined from Sca tchard a n a l y s i s , of the h e p a t i c HALC ABP; and 3) to attempt to r e v e r s e the changes seen i n the HALC ABP of STZ d i a b e t i c male r a t s by t reatment with 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 oGH. MATERIALS AND METHODS I . Chemica ls The f o l l o w i n g chemica l s were purchased from Sigma Chemical Company ( S t . L o u i s , MO): T r i s (hydroxymethyl)aminomethane (Trizma®-base), e t h y l e n e d i a m i n e - t e t r a a c e t i c a c i d , D L - d i t h i o t h r e i t o l , sodium molybdate , a c t i v a t e d c h a r c o a l , bovine serum a lbumin , B r i l l a n t b lue G 250, s t r e p t o z o t o c i n ( S T Z ) , c i t r i c a c i d ( f r e e a c i d and t r i s o d i u m s a l t ) , t e s t o s t e r o n e e n a n t h a t e , t r i a m c i n o l o n e a c e t o n i d e , 5a-DHT, 58-DHT, a n d r o s t e n e d i o n e , e s t r a d i o l , d i e t h y l s t i 1 b e s t e r o l , p r o g e s t e r o n e , and t r i i odothyron i ne. 3 R a d i o a c t i v e l y l a b e l e d [ 1 7 a - m e t h y l - H] m e t h y l t r i e n o l o n e (R1881, s p e c i f i c a c t i v i t y 86 or 87 Ci/mmol) and R1881 were purchased from New England N u c l e a r , under l i c e n c e d agreement wi th R o u s s e l - U c l a f ( R o m a i n v i l l e , F r a n c e ) . Biofluor® was purchased from New England N u c l e a r . Ovine growth hormone (oGH) was p rov ided by Dr . S. R a i t i ( N a t i o n a l Hormone and P i t u i t a r y Program, B a l t i m o r e M a r y l a n d ) . Dextran T70® was purchased from Pharmacia F ine Chemica ls ( U p p s a l a , Sweden). G l y c e r o l was purchased from B r i t i s h Drug House C h e m i c a l s . Ammonium s u l f a t e , u l t r a p u r e , was ob ta ined from ICN B i o m e d i c a l . PZI and Toronto i n s u l i n , beef and pork , (100 U/mL) from Connaught L a b o r a t o r i e s ( W i l l o w d a l e , O n t a r i o ) were used . S i l i c a gel t h i n l a y e r chromatography sheets (20 x 20 cm) wi th f l u o r s c e n t i n d i c a t o r were purchased from Eastman Kodak Co. (Rochester NY). Osmotic pumps were purchased from A l z e t C o r p o r a t i o n (Pa lo A l t o , C a l i f o r n i a ) . S o l v e n t s and o ther reagents used were of reagent grade . Rad iochemica l 3 p u r i t y of [ H]R1881 was v e r i f i e d by t h i n l a y e r chromatography. Two s o l v e n t systems were used , benzene : e t h y l a c e t a t e ( 8 : 2 ) , and c h o l o r o f o r m : e t h a n o l ( 9 8 : 2 ) . Checks were performed on each new shipment and p e r i o d i c a l l y t h e r e a f t e r . Rad iochemica l p u r i t y of 97% or above was c o n s i d e r e d a c c e p t a b l e . I I . Care and surgery of animals A d u l t male (240-390 g ) , a d u l t female (200-260 g ) , and pregnant female Wis ta r r a t s were purchased from Canada Breed ing L a b o r a t o r i e s (La P r a i r i e , Quebec) . Animals were housed, 4 to a cage , in p l a s t i c cages on corncob bedd ing , Lobund Grade (Paxton P r o c e s s i n g L t d . Paxton , I I . Lobund grade bedding i s p r e s e r v a t i v e f r e e ) , at 22°C, 14 hours l i g h t (0600-2000) , 10 hours dark . Pur ina Lab Chow and tap water were f r e e l y a v a i l a b l e . A d u l t male Wis ta r r a t s were made d i a b e t i c under e ther a n a e s t h e s i a by a s i n g l e t a i l ve in i n j e c t i o n of STZ, a d m i n i s t e r e d in 0.1 M c i t r a t e b u f f e r , pH = 4 . 5 , at a dose of 60 mg/kg. Exper iments were done us ing 2 1 both 4 and 10 day p o s t - i n j e c t i o n d i a b e t i c r a t s . A l l r a t s i n j e c t e d with STZ, with the e x c e p t i o n of those t r e a t e d with i n s u l i n , had b lood g lucose l e v e l s of 0.5 g/100 mL or g r e a t e r , as determined by TesTape® ( E l i L i l l y and Company L t d . , T o r o n t o , O n t a r i o ) u r i n e measurements. Neonatal c a s t r a t i o n s were performed by the abdominal route us ing i c e a n a e s t h e s i a ( G u s t a f s s o n and S t e n b e r g , 1974) . Minipumps were imp lanted under e ther a n a e s t h e s i a subcutaneous ly i n t o the d o r s a l r e g i o n . Vena cava c a t h e t e r s were imp lanted by the abdominal route under e t h e r or ha lothane a n a e s t h e s i a , as d e s c r i b e d in the r e s u l t s . L o c a l a n a e s t h e t i c ( l i d o c a i n e ) and t o p i c a l a n t i b i o t i c ( C i c a t r i n ®, each g c o n t a i n s : neomycin s u l p h a t e BP 3300 IU, Z inc B a c i t r a c i n BP 250 I . U . , L - c y s t e i n e 2 mg, . D L - t h r e o n i n e 1 mg, and g l y c i n e 10 mg) were used at the i n c i s i o n and s i t e of c a t h e t e r sk in e n t r y . I I I . Treatment of an imals The replacement doses of i n s u l i n were: PZI , 10 U/kg; T o r o n t o , 15 U/kg ( O r t i z - C a r o et al , 1984). PZI was a d m i n i s t e r e d once a day; Toronto i n s u l i n , twice a day. Exper iments were done 24 or 12 hours , r e s p e c t i v e l y , f o l l o w i n g the l a s t i n s u l i n dose. The rep lacement dose of t e s t o s t e r o n e enanthate used in STZ d i a b e t i c animals was 2.5 nmol/kg in corn o i l . Th i s dose was chosen because i t has been shown to produce p h y s i o l o g i c a l serum l e v e l s in c a s t r a t e d r a t s (Sunahara , 1984) . Exper iments were done 12 or 24 hours a f t e r the l a s t dose of t e s t o s t e r o n e e n a n t h a t e . T r i i o d o t h y r o n i n e was a d m i n i s t e r e d in a dose of 30 ng/kg d a i l y ( T a h a l i a n i , 1983) . The dose of growth hormone s e c r e t e d by minipump was 0.02 U/hour, f o r four days . The dose of growth hormone i n the 7 d a i l y s . c . dose exper iment , the 7 d a i l y doses a d m i n i n s t e r e d by vena cava c a t h e t e r , and the 4 d a i l y t a i l ve in i n j e c t i o n s was 30 ug per r a t per dose , a d m i n i s t e r e d in s a l i n e (pH 8 . 5 ) . IV. P r e p a r a t i o n of c y t o s o l f o r b i n d i n g assays 1. P r e p a r a t i o n of whole c y t o s o l An ima ls were k i l l e d by s t u n n i n g , c e r v i c a l d i s l o c a t i o n , then d e c a p i t a t i o n . L i v e r s were pe r fused with i c e c o l d TEDM b u f f e r (0 .05 M T r i s - b a s e ; 1.5 mM e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d d i s o d i u m ; 0.5 mM d i t h i o t h r e i t o l , 10% g l y c e r o l ; 20 mM sodium m o l y b d a t e ) , and were d i s s e c t e d , we ighed , minced , then homogenized, 1:15 (w/v) w i th f r e s h TEDM b u f f e r , at 4°C u s i n g a p r e c o o l e d P o t t e r - E 1 v e j h e i m homogenizer with t e f l o n p e s t l e . The crude homogenate was c e n t r i f u g e d at 10,000 x g f o r 10 minutes at 4°C in an I n t e r n a t i o n a l Equipment Company, model B-20 c e n t r i f u g e . Th i s crude c y t o s o l was c e n t r i f u g e d at 105,000 x g f o r 65 minutes at 4°C i n Beckman model L5 -50 , or L2-65B u l t r a c e n t r i f u g e s . Whole c y t o s o l was d i l u t e d by 2 3 o n e - h a l f wi th TEDM b u f f e r to produce 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, and was then incubated (see below, V, 1 ) . 2. F r a c t i o n a t i o n of whole c y t o s o l Whole c y t o s o l prepared as above was f r a c t i o n a t e d with ammonium s u l f a t e a c c o r d i n g to the f o l l o w i n g p r o c e d u r e . S a t u r a t e d ammonium s u l f a t e s o l u t i o n was added drop by drop over h a l f an hour to whole c y t o s o l which was s t i r r i n g on i c e . T h i s was c e n t r i f u g e d at 12,000 x g f o r 30 m i n u t e s . The p e l l e t was resuspended with TEDM b u f f e r , and incuba ted i n the same manner as the whole c y t o s o l (see below, p a r t V, 1 ) . 3. F r e e z i n g of the 50 % ammonium s u l f a t e f r a c t i o n a t e d c y t o s o l i c p e l l e t P e l l e t s from f r a c t i o n a t e d c y t o s o l were quick f r o z e n i n ace tone-dry i c e , then s t o r e d at -80°C f o r p e r i o d s up to 60 days , in o r d e r to determine the s t a b i l i t y of the HALC ABP to f r e e z i n g . P e l l e t s were resuspended in a volume of i c e c o l d TEDM b u f f e r equal to the volume of o r i g i n a l c y t o s o l immediate ly be fore the i n c u b a t i o n was s t a r t e d . 4. P r o t e i n c o n c e n t r a t i o n d e t e r m i n a t i o n P r o t e i n c o n c e n t r a t i o n i n whole and f r a c t i o n a t e d c y t o s o l 2 4 was determined by the method of B rad fo rd (1976) , us ing a P e r k i n Elmer Model 124 double beam s p e c t r o p h o t o m e t e r . The s tandard curve was generated us ing bovine serum albumin in c o n c e n t r a t i o n s of 0.10 to 0.40 mg/mL. V. R a d i o l i g a n d exchange b i n d i n g s t u d i e s 1. Incubat ion of c y t o s o l Cy toso l was incubated a c c o r d i n g to the method of Sunahara ( 1 9 8 4 ) . For S c a t c h a r d a n a l y s i s a 500 uL a l i q u o t of c y t o s o l ( 0 . 5 - 1 . 0 mg prote in/mL) was incuba ted with 3 i n c r e a s i n g c o n c e n t r a t i o n s of [ H]R1881 i n the p r e s e n c e and absence of 100 f o l d molar excess R1881, and with 100 f o l d molar excess of t r i a m c i n o l o n e a c e t o n i d e . T h i s mixture was i n c u b a t e d at 4°C f o r 3 h o u r s . In s p e c i f i c i t y s t u d i e s , a 500 a l i q u o t of c y t o s o l ( 0 . 5 - 1 . 0 mg prote in/mL) was i n c u b a t e d with 1 nM [ 3 H]R1881 and 0.1 to 1000 f o l d molar excess of R1881, as wel l as 100 f o l d molar excess of 1 nM t r i a m c i n o l o n e a c e t o n i d e . When R1881 was used as the c o m p e t i t o r , lower c o n c e n t r a t i o n s of R1881 (0.0001 to 1000 f o l d molar excess) were used to generate a complete d i sp lacement c u r v e . 2 5 2. S e p a r a t i o n of bound and f r e e s t e r o i d f r a c t i o n s Free s t e r o i d was removed from bound us ing 2.5% d e x t r a n - c o a t e d charcoa l in TEDM b u f f e r as d e s c r i b e d by Beato and F e i g e l s o n (1972) and C la rk and Peck (1980) . T h i s c o n t a i n e d 2.5% c h a r c o a l i n TEDM b u f f e r pH 7 . 5 , with 0.25% Dextran T70®. A l i q u o t s of 500 M-L were added to each i n c u b a t i o n t u b e , tubes were v o r t e x e d l i g h t l y and c e n t r i f u g e d at 1500 x g f o r 10 minutes at 4°C. F i v e hundred nL of the supernant was added to 10 mL Biof luor®. L i q u i d s c i n t i l l a t i o n c o u n t i n g was done in a Mark I II l i q u i d s c i n t i l l a t i o n counte r 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 e s , II 60018) . T r i t i u m was quenched 35 to 47%. Data was recorded onto a microcomputer d i s k e t t e u s i n g an Apple I I - p l u s d i s c - o p e r a t i n g system (Apple Computer I n c . , C u p e r t i n o , CA 95014), e i t h e r connected d i r e c t l y to the Mark III by a s e r i a l ca rd i n t e r f a c e or entered manua l l y . V I . A n a l y s i s of r a d i o a c t i v i t y Free l i g a n d c o n c e n t r a t i o n was determined by 3 s u b t r a c t i n g counts due to s p e c i f i c b i n d i n g of [ H]R1881 from t o t a l r a d i o a c t i v i t y added to each tube . Bound r a d i o a c t i v i t y was the r a d i o a c t i v i t y in i n c u b a t i o n tubes with r a d i o l a b e l e d l i g a n d and t r i a m c i n o l o n e a c e t o n i d e o n l y ; n o n - s p e c i f i c bound was determined from the r a d i o a c t i v i t y in i n c u b a t i o n tubes with r a d i o l a b e l e d R1881, 100 f o l d molar excess l i g a n d and t r i a m c i n o l o n e a c e t o n i d e . S p e c i f i c bound was determined by s u b t r a c t i n g n o n - s p e c i f i c bound from bound. B i n d i n g was expressed as fmol/mg p r o t e i n , and was ana lysed us ing l i n e a r Sca t chard p l o t s ( s p e c i f i c bound/free versus s p e c i f i c bound; S c a t c h a r d , 1949). B i n d i n g k i n e t i c s were expressed as apparent d i s s o c i a t i o n c o n s t a n t of e q u i l i b r i u m (K^; nM) and a p p a r e n t maximum number of b i n d i n g s i t e s or b i n d i n g c a p a c i t y ( B m a x ; fmol/mg of p r o t e i n ) . V I I . S t a t i s t i c s ANOVA and Newman-Keul's Range t e s t were used to determine s i g n i f i c a n c e of d i f f e r e n c e s . The n u l l h y p o t h e s i s was assumed and the l e v e l of s i g n i f i c a n c e was P < 0 . 0 5 . RESULTS I . The e f f e c t s of ammonium s u l f a t e f r a c t i o n a t i o n of c y t o s o l on the h e p a t i c high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n 1. The e f f e c t of ammonium s u l f a t e f r a c t i o n a t i o n on the Kd and b i n d i n g c a p a c i t y va lues of the high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n Ammonium s u l f a t e f r a c t i o n a t i o n was at tempted i n o r d e r to p a r t i a l l y p u r i f y the HALC ABP. Th i s was d e s i r a b l e to decrease n o n - s p e c i f i c b i n d i n g and t h e r e f o r e to more r e a d i l y see changes in the amount of HALC ABP in d i f f e r e n t p h y s i o l o g i c a l s t a t e s such as d i a b e t e s . P r e l i m i n a r y exper iments in which 50 % ammonium s u l f a t e f r a c t i o n a t i o n was 2 used r e l i a b l y r e s u l t e d in l i n e a r S c a t c h a r d p l o t s (r 0.80 or above) so to determine the percentage of ammonium s u l f a t e r e q u i r e d f o r p u r i f i c a t i o n , exper iments u s i n g a range of c o n c e n t r a t i o n s were done. A l so a two step procedure was used in which an i n i t i a l p r e c i p i t a t i o n with 33 % ammonium s u l f a t e was d i s c a r d e d , and a second p e l l e t , p repared by p r e c i p i t a t i o n with e i t h e r a f u r t h e r 17 or 32 % ammonium s u l f a t e ( f i n a l amount of ammonium s u l f a t e 50 or 65 %), was resuspended and used in the i n c u b a t i o n . R e s u l t s from 33, 50, 65 % and from the two step procedure are in Tab le I . Tab le I THE EFFECT OF AMMONIUM SULFATE FRACTIONATION OF WHOLE HEPATIC CYTOSOL ON THE HIGH AFFINITY-LOW CAPACITY ANDROGEN BINDING PROTEIN ASSAY Standard assay c o n d i t i o n s were used except tha t d i f f e r e n t amounts of ammonium s u l f a t e were used to p a r t i a l l y p u r i f y the h e p a t i c c y t o s o l . % F r a c t i o n a t i o n Kd (nM) Bi ndi ng capac i ty (fmol/mg) Whole Cy toso l 0.32 + 0. 04 5.86 + 0.67 (17) 33 1.01 * 82.91 * 2/6 ** 50 0.30 + 0. 02 10.37 + 2.34 5/6 ** 65 0.33 + 0. 07 6.40 + 1.15 6/6 ** 33-50 0.13 8.55 1/4 ** 33-65 0.13 + 0. 07 4.30 + 0.37 3/4 ** * I n d i c a t e s va lues s i g n i f i c a n t l y d i f f e r e n t from a l l o ther va lues at the P < 0.05 l e v e l us ing ANOVA and Newman-Keul's Range t e s t ** the denominator r e p r e s e n t s the number of assays done, the numerator , the number of l i n e a r Sca t chard p l o t s ob ta ined From the t a b l e i t can be seen tha t 33 % f r a c t i o n a t i o n d i d not r e l i a b l y produce l i n e a r S c a t c h a r d p l o t s , and produced a Kd s i g n i f i c a n t l y d i f f e r e n t from the Kd of the HALC ABP p r e v i o u s l y shown to have h igh androgen s p e c i f i c i t y in the whole c y t o s o l . The two step f r a c t i o n a t i o n procedure proved d i s a p p o i n t i n g as i t was r a r e l y produced l i n e a r S c a t c h a r d p l o t s . F i f t y to s i x t y - f i v e percent ammonium s u l f a t e f r a c t i o n a t i o n produced l i n e a r S c a t c h a r d p l o t s r e l i a b l y , and there were no major d i f f e r e n c e s in Kd va lues compared to u n f r a c t i o n a t e d c y t o s o l . The d e c i s i o n to c o n t i n u e wi th a 50 % f r a c t i o n a t i o n was based on t h i s and o ther p r e l i m i n a r y data c o l l e c t e d , in which 50 % ammonium s u l f a t e f r a c t i o n a t i o n had been used , and had produced r e l i a b l e and c o n s i s t e n t r e s u l t s . F i f t y p e r c e n t ammonium s u l f a t e f r a c t i o n a t i o n r e s u l t e d i n a 2 to 3 f o l d p u r i f i c a t i o n based on i n c r e a s e d b i n d i n g c a p a c i t y ( t a b l e s I and I I I ) . 2. The presence of the g l u c o c o r t i c o i d r e c e p t o r in the 50 % ammonium s u l f a t e f r a c t i o n a t e d h e p a t i c c y t o s o l of the a d u l t male r a t Three exper iments were done in which the n e c e s s i t y of add ing t r i a m c i n o l o n e a c e t o n i d e to p a r t i a l l y p u r i f i e d c y t o s o l was examined. Th i s was to determine whether or not the g l u c o c o r t i c o i d r e c e p t o r c o - p u r i f i e d with the HALC ABP. A r e p r e s e n t a t i v e s a t u r a t i o n b i n d i n g curve and S c a t c h a r d p l o t 30 F i g u r e 1 SATURATION CURVES AND SCATCHARD PLOTS FOR BINDING STUDIES OF THE HEPATIC CYTOSOLIC HIGH AFFINITY-LOW CAPACITY ANDROGEN BINDING PROTEIN IN 50 % AMMONIUM SULFATE FRACTIONATED CYTOSOL WITH AND WITHOUT TRIAMCINOLONE ACETONIDE A d u l t (240-390 g) male r a t h e p a t i c c y t o s o l was prepared and f r a c t i o n a t e d with 50 % ammonium s u l f a t e as d e s c r i b e d in the Methods s e c t i o n and was incubated with v a r y i n g c o n c e n t r a t i o n s of [ 3 H]R1881 (0 .05-5 nM) with or wi thout 100 f o l d molar excess R1881, and wi th (a) or w i thout (b) 100 f o l d molar excess t r i a m c i n o l o n e a c e t o n i d e , f o r 3 hours at 4°C . a: S c a t c h a r d a n a l y s i s and, i n s e t , s a t u r a t i o n curve w i thout t r i a m c i n o l o n e aceton ide b: S c a t c h a r d a n a l y s i s and, i n s e t , s a t u r a t i o n curve with t r i a m c i n o l o n e aceton ide F I G U R E 1 b S p e c i f i c Bound (fmol/mg) i s seen in f i g u r e 1, a and b. Ev idence of 2 b i n d i n g s i t e s may be seen in b o t h , t h e r e f o r e we c o n t i n u e d the i n c l u s i o n of t r i a m c i n o l o n e in the i n c u b a t i o n m i x t u r e . 3. The e f f e c t of 50 % ammonium s u l f a t e f r a c t i o n a t i o n on the optimum i n c u b a t i o n time f o r the h igh a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n assay In order to determine the optimum i n c u b a t i o n time f o r the HALC ABP b i n d i n g a s s a y , exper iments u s i n g whole and p a r t i a l l y p u r i f i e d h e p a t i c c y t o s o l were conducted f o r time p e r i o d s from 1.5 to 30 h o u r s . A s i m i l a r exper iment was c a r r i e d out with p a r t i a l l y p u r f i e d h e p a t i c c y t o s o l from 10 day STZ d i a b e t i c r a t s . R e s u l t s are shown in t a b l e II ( 1 - 3 ) . A l though there i s some v a r i a t i o n in the d a t a , a f t e r 3 hours b i n d i n g c h a r a c t e r i s t i c s of the HALC ABP d id not change s i g n i f i c a n t l y in whole or in f r a c t i o n a t e d h e p a t i c c y t o s o l . I I . The e f f e c t of the p e r f u s a t e volume on the r e s u l t s of the h igh a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n assay E a r l y in our s t u d i e s the sugges t ion tha t b lood c o n t a m i n a t i o n of h e p a t i c c y t o s o l c o u l d produce a r t i f a c t u a l r e s u l t s due to l a r g e amounts of n o n - s p e c i f i c p o l y c y c l i c aromat ic hydrocarbon b i n d i n g p r o t e i n s ( P o e l l i n g e r et a l , 3 4 Tab le II THE EFFECT OF INCUBATION TIMES FROM 1.5 TO 30 HOURS ON THE Kd AND BINDING CAPACITY VALUES OF THE HEPATIC CYTOSOLIC HIGH AFFINITY-LOW CAPACITY ANDROGEN BINDING PROTEIN 1. Whole c y t o s o l from c o n t r o l r a t s S tandard assay c o n d i t i o n s were used except tha t i n c u b a t i o n t imes of 1.5 to 30 hours were used . T i me(hours) Kd (nM) Bi ndi ng Capaci ty (f mol /mgT 1.5 1.07 6.67 3 0.17 1.97 6 0.09 2.43 9 0.23 2.76 12 0.16 2.64 15 0.16 2.29 18 0.09 7.10 21 0.12 3.43 24 0.35 5.25 30 0.08 2.23 Table II 2. 50 % ammonium s u l f a t e f r a c t i o n a t e d c y t o s o l from c o n t r o l r a t s Time (hours) Kd (nM) B ind ing C a p a c i t y K[ (f mol /mgT 1.5 0.46 + 0.13 25.53 + 6.61 5 3 0.30 + 0.02 21.03 + 1.49 43 * 6 0.29 + 0.09 20.93 + 7.43 5 9 0.42 + 0.12 25.31 + 12.50 5 18 0.23 + 0.15 27.09 + 6.04 2 21 0.17 + 0.09 22.43 + 1.56 2 24 0.10 + 0.01 18.69 + 3.86 2 30 0.23 + 0.12 29.51 + 11.78 2 Values are not s i g n i f i c a n t l y d i f f e r e n t from each other at (P < 0.05) us ing ANOVA and Newman-Keul 1s Range t e s t . * Unt reated c o n t r o l s were assayed in exper iments p resented in the f o l l o w i n g t a b l e s : I I , I I I , IV, V, VI , V I I , and X. 3 6 Tab le II 3. 50 % ammonium s u l f a t e f r a c t i o n a t e d c y t o s o l from 10 day s t r e p t o z o t o c i n d i a b e t i c r a t s Time (hours) Kd (nM) B i n d i n g C a p a c i t y (fmol/mg) 1.5 0.27 13.36 3 0.30 15.90 6 0.20 9.43 9 0.23 11.39 18 0.22 14.26 21 0.14 8.05 24 0.37 16.60 27 0.55 25.72 30 0.12 9.42 Tab le III THE EFFECT OF DIFFERENT LIVER PERFUSION VOLUMES USED PRIOR TO HIGH AFFINITY-LOW CAPACITY ANDROGEN BINDING PROTEIN HALC ABP ASSAYS DONE IN WHOLE AND 50 % AMMONIUM SULFATE FRACTIONATED HEPATIC CYTOSOL L i v e r s were pe r fused with 10 or 100 mL of TEDM b u f f e r , then s tandard assay c o n d i t i o n s were used . Type of P e r f u s i o n Kd B i n d i n g C a p a c i t y Cy toso l Volume (mL) (nM) (fmol /mgT whole 10 0.37 5.72 a 50 % ammonium 10 0.38 36.95 a s u l f a t e f r a c t i o n a t e d whole 100 0.31 4.93 b 50 % ammonium 100 0.05 3.95 b s u l f a t e f r a c t i o n a t e d whole 10 0.31 7.48 c 50 % ammonium 10 0.32 20.41 c s u l f a t e f r a c t i o n a t e d whole 100 0.49 10.16 d 50 % ammonium 100 0.37 29.41 d s u l f a t e f r a c t i o n a t e d L e t t e r s i n d i c a t e samples taken from the same l i v e r 3 8 1983). In order to i n v e s t i g a t e t h i s p o s s i b i l i t y , exper iments us ing l i v e r s p e r f u s e d with 10 or 100 mL of TEDM b u f f e r were done. Assays were performed on whole and p a r t i a l l y p u r i f i e d c y t o s o l . R e s u l t s are p resented in t a b l e I I I and show no major d i f f e r e n c e s i n Kd and b i n d i n g c a p a c i t y between samples homogenized from l i v e r p e r f u s e d with e i t h e r 10 or 100 mL TEDM b u f f e r . One 50 % ammonium s u l f a t e f r a c t i o n a t e d sample showed a b i n d i n g c a p a c i t y much decreased from other ammonium s u l f a t e p u r i f i e d c y t o s o l volumes, however, t h i s sample a l s o e x i b i t e d an unusual Kd v a l u e . Exper iments in which b lood was used in p lace of h e p a t i c c y t o s o l in the HALC ABP assay were a l s o done. In 4 exper iments no d i s p l a c a b l e b i n d i n g was e v i d e n t . I I I . E f f e c t of f r e e z i n g on the high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n P r e l i m i n a r y s t u d i e s were done to examine f r o z e n s to rage of the HALC ABP. The HALC ABP cou ld be p r e s e r v e d f r o z e n at -80°C in the p e l l e t prepared with 50 % ammonium s u l f a t e . Three S c a t c h a r d p l o t s c o n s t r u c t e d from data ob ta ined from the p a r t i a l l y p u r i f i e d h e p a t i c c y t o s o l of one a n i m a l , assayed at t h r e e t ime p o i n t s : on the day of k i l l , and on one day and two months f o l l o w i n g are in f i g u r e 2. Kd va lues f o r 0, 1, and 60 days of s to rage a r e , r e s p e c t i v e l y , 0 .15 , 0 .15 , and 0.25 nM; va lues f o r b i n d i n g c a p a c i t y at the same time 39 F i g u r e 2 SCATCHARD PLOT OF THE HEPATIC CYTOSOLIC HIGH AFFINITY-LOW CAPACITY ANDROGEN BINDING PROTEIN FROM FROZEN 50 % AMMONIUM SULFATE PELLET A d u l t (240-390 g) male r a t h e p a t i c c y t o s o l f r a c t i o n a t e d with 50 % ammonium s u l f a t e ( 0 . 0 5 - 1 . 0 mg/mL) was prepared to the ammonium s u l f a t e p e l l e t s t a g e , as d e s c r i b e d in the Methods s e c t i o n . The ammonium s u l f a t e p e l l e t was quick f r o z e n i n dry i c e - a c e t o n e , then s t o r e d at -80°C u n t i l use . S torage time was e i t h e r 0, 1, or 60 days . The resuspended p e l l e t was i n c u b a t e d wi th v a r y i n g c o n c e n t r a t i o n s of t r i t i a t e d R1881 (0 .05-5 nM), with or w i thout 100 f o l d molar excess R1881, and wi th 100 f o l d molar excess t r i a m c i n o l o n e a c e t o n i d e f o r 3 hours at 4°C. 4 0 F I G U R E 2 p o i n t s a r e , r e s p e c t i v e l y , 3 3 . 3 , 3 0 . 6 , and 30.0 fmol/mg p r o t e i n . E s s e n t i a l l y no changes in Kd or in b i n d i n g c a p a c i t y were seen in our assay , IV. Age and sex d i f f e r e n c e s in the presence of the h e p a t i c c y t o s o l i c high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n in r a t s 1. In mature female r a t s The r e s u l t s of exper iments done in mature female W i s t a r r a t s are in t a b l e IV. There was l e s s than o n e - h a l f the amount of h e p a t i c c y t o s o l i c HALC ABP in female as in male r a t s , when 100 f o l d molar excess t r i a m c i n o l o n e a c e t o n i d e was used in b o t h . However, t h i s may not be a c o r r e c t i n t e r p r e t a t i o n because when t r i a m c i n o l o n e a c e t o n i d e was r i n c r e a s e d to 500 f o l d molar e x c e s s , there was an a p p a r e n t , a l though not s t a t i s t i c a l l y s i g n i f i c a n t , decrease in b i n d i n g c a p a c i t y in f ema les , but no change in the b i n d i n g c a p a c i t y of m a l e s . There i s a s i g n i f i c a n t d i f f e r e n c e in the Kd va lue between the HALC ABP of males and females with 100 versus 500 f o l d molar excess t r i a m c i n o l o n e a c e t o n i d e , and a s i g n i f i c a n t d i f f e r e n c e between the Kd of the HALC ABP males versus females with 100 or 500 f o l d molar excess t r i a m c i n l o n e a c e t o n i d e . Tab le IV THE EFFECT OF INCREASING AMOUNTS OF TRIAMCINOLONE ACETONIDE IN ASSAYS OF MALE AND FEMALE RAT HEPATIC CYTOSOLIC HIGH AFFINITY-LOW CAPACITY ANDROGEN BINDING PROTEIN Standard assay c o n d i t i o n s were used wi th male and female h e p a t i c c y t o s o l . D i f f e r e n t amounts of t r i a m c i n o l o n e a c e t o n i d e were i n c l u d e d in the i n c u b a t i o n m i x t u r e . Animal Model Kd (nM) Bi "d ing Capaci ty N^  (fmol / m g T sex amount of t r i amci no!one  molar excess male 100 f o l d 0.30 + 0.02a 20.76 + 1.49d 43 male 500 f o l d 0.48 + 0.10b 16.65 + 6.91d 5 male 1000 f o l d 0.62 19.08 1/2 * female female female female 0 100 f o l d 500 f o l d 1000 f o l d 0.37 0 . 1 5 + 0.03c 0.21 + 0 .03a , c 0.04 16.00 1/8 * 7.04 + 0.94e 14 3.00 + 0.13e 8 2.05 1/6 * S i m i l a r l e t t e r s i n d i c a t e no s i g n i f i c a n t d i f f e r e n c e at P < 0.05 l e v e l us ing ANOVA and Newman-Keul's range t e s t . * the denominator i n d i c a t e s the number of h e p a t i c HALC ABP assays done, the numerator i n d i c a t e s the number which produced l i n e a r Sca t chard p l o t s 2. In mature male and female r a t s Exper iments in which poo ls of l i v e r from immature male and female r a t s were used showed equal b i n d i n g c a p a c i t y HALC ABP i n b o t h . T h i s b i n d i n g c a p a c i t y i s s i m i l a r to the t h a t of the mature female (see t a b l e V ) . Exper iments us ing i n c r e a s i n g amounts of t r i a m c i n o l o n e a c e t o n i d e were not done in immature male or female r a t s . 3. In c a s t r a t e d male r a t s Tab le VI shows the r e s u l t s of HALC ABP assays done on c a s t r a t e d mature males one week f o l l o w i n g c a s t r a t i o n . There i s no s i g n i f i c a n t d i f f e r e n c e a c c o r d i n g to ANOVA and Newman-K e u l ' s Range t e s t in e i t h e r Kd or b i n d i n g c a p a c i t y va lues between c o n t r o l , sham and c a s t r a t e d mature males . Neonatal c a s t r a t i o n s were performed on male r a t s which were then a l lowed to grow to m a t u r i t y . A l though s p e c i f i c b i n d i n g was s t i l l p resent in n e o n a t a l l y c a s t r a t e d r a t s , S c a t c h a r d p l o t s were ob ta ined f o r only 2 of 6 r a t s , and those had very low b i n d i n g c a p a c i t i e s (3 .74 and 2.76 fmol/mg p r o t e i n ) . Rats c a s t r a t e d n e o n a t a l l y , then t r e a t e d wi th t e s t o s t e r o n e enanthate i n a dose of 17 mmol/kg at day 8 (S loop et al , 1983) showed low b i n d i n g c a p a c i t i e s (5.81 + 2.04 fmol/mg), but c o n s i s t e n t S c a t c h a r d p l o t s . 4 4 Tab le V THE HEPATIC HIGH AFFINITY-LOW CAPACITY ANDROGEN BINDING PROTEIN CONTENT OF MATURE AND IMMATURE MALE AND FEMALE RATS Standard assay c o n d i t i o n s were used with h e p a t i c c y t o s o l from A d u l t (240-390 g) male, a d u l t (200-260 g) female and and immature (30-40 g) male and female r a t . Animal Model Kd (nM) B ind ing C a p a c i t y (fmol/mg) mature male 0.30 + 0.02a 20.76 + 1.49c 43 immature male 0.10 9.45 pool 1 0.15 5.40 pool 2 mature female 0.15 + 0.03b 7.04 + 0.94d 14 immature female 0.40 9.20 pool 1 0.07 9.57 pool 2 S i m i l a r l e t t e r s i n d i c a t e no s i g n i f i c a n t d i f f e r e n c e (P < 0.05) us ing ANOVA and Newman-Keul's range t e s t Table VI THE EFFECT OF CASTRATION ON THE PRESENCE OF THE HEPATIC CYTOSOLIC HIGH AFFINITY-LOW CAPACITY ANDROGEN BINDING PROTEINS IN MALE RATS Standard assay c o n d i t i o n s were used with h e p a t i c c y t o s o l from c a s t r a t e d a d u l t (240-390 g) male r a t s , sham c a s t r a t e d a d u l t male r a t s , n e o n a t a l l y c a s t r a t e d a d u l t male r a t s , and n e o n a t a l l y c a s t r a t e d t e s t o s t e o n e t r e a t e d (17 mmol/kg, day 8) a d u l t male r a t s . Animal Model Kd (nM) Bi nding Capaci ty ( fmol / m g T male 0.30 + 0.02 20.76 + 1.49 (43) mature sham 0.18 + 0.04 10.90 + 4.10 (4) c a s t r a t e d male mature 0.33 + 0.08 11.85 + 2.73 (6) c a s t r a t e d male n e o n a t a l l y c a s t r a t e d male 0.77 3.74 n e o n a t a l l y c a s t r a t e d male 0.10 2.76 2/6 * n e o n a t a l l y 0.06 + 0.02 5.81 + 2.04 3/3 * c a s t r a t e d t e s t o s t e r o n e t r e a t e d mal e ( ) = N * the denominator r e p r e s e n t s t o t a l number of assays done, the numerator , the number of assays r e s u l t i n g in l i n e a r S c a t c h a r d p l o t s ANOVA and Newman-Keul's range t e s t showed no s i g n i f i c a n t d i f f e r e n c e between male and c a s t r a t e d male Kd and b i n d i n g c a p a c i t i e s . S t a t i s t i c s were not done on neonatal c a s t r a t e d r a t s because of the low N v a l u e . V. Presence of the high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n in the h e p a t i c c y t o s o l of g e n e t i c a l l y d i a b e t i c , B ioBreed ing s t r a i n , r a t s Dur ing t h i s study i t was p lanned tha t g e n e t i c a l l y d i a b e t i c r a t s ( B i o B r e e d i n g s t r a i n ) would be compared wi th STZ induced d i a b e t i c s . L i m i t e d access due to co lony problems r e s u l t e d in the assay of only three male B i o B r e e d i n g s t r a i n d i a b e t i c s and f i v e male B i o B r e e d i n g n o n - o v e r t l y d i a b e t i c l i t t e r m a t e c o n t r o l s . Presence of d i a b e t e s was determined by the presence of g l u c o s u r i a . R e s u l t s from these exper iments are in t a b l e V I I . No d i f f e r e n c e s were seen in Kd v a l u e s . The b i n d i n g c a p a c i t y of n o n - d i a b e t i c and d i a b e t i c B ioBreed ing s t r a i n r a t s were s i g n i f i c a n t l y reduced from Wis tar c o n t r o l v a l u e s . No s i g n i f i c a n t d i f f e r e n c e was seen in b i n d i n g c a p a c i t y between d i a b e t i c and n o n - d i a b e t i c B i o B r e e d i n g s t r a i n r a t s . I t would be worthwhi le to i n c r e a s e numbers of animals in order to v e r i f y whether an i n c r e a s e d number would prove the apparent d i f f e r e n c e s i g n i f i c a n t . Tab le VII BINDING PARAMETERS IN BIOBREEDING STRAIN DIABETIC, AND BIOBREEDING STRAIN NON-DIABETIC MALE RATS COMPARED TO CONTROL ADULT MALE RATS Standard assay c o n d i t i o n s were used with h e p a t i c c y t o s o l from A d u l t male B i o B r e e d i n g s t r a i n d i a b e t i c (280-320 g ) , B i o B r e e d i n g - s t r a i n n o n - d i a b e t i c (390-440 g) and Wis ta r c o n t r o l r a t (240-390 g) Animal Model Kd (nM) B i n d i n g Capaci ty (fmol/mg) Wis ta r c o n t r o l 0.30 + 0.02 20.76 + 1.49 a (43) B i o B r e e d i n g s t r a i n 0.36 + 0.04 11.57 + 2.23 b (5) i n t e r n a l c o n t r o l B i o B r e e d i n g s t r a i n 0.49 + 0.32 4.58 + 0.73 b (3) d i a b e t i c ( ) = N S i m i l a r l e t t e r s i n d i c a t e no s i g n i f i c a n t d i f f e r e n c e at the < 0.05 l e v e l us ing ANOVA and Newman-Keul's Range t e s t 4 8 VI . E f f e c t s of s t r e p t o z o t o c i n induced d i a b e t e s on the Kd v a l u e s , b i n d i n g c a p a c i t y , temperature s t a b i l i t y , and s t e r o i d s p e c i f i c i t y of the high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n 1. High a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n b i n d i n g s t u d i e s in c o n t r o l , 4 and 10 day s t r e p t o z o t o c i n d i a b e t i c r a t s P r e l i m i n a r y s t u d i e s in our l a b o r a t o r y (Sunahara , 1984) showed tha t a decrease in b i n d i n g c a p a c i t y of the HALC ABP was seen in the l i v e r of r a t s four days a f t e r i n d u c t i o n of d i a b e t e s with STZ. In o rder to determine i f there were o ther changes in the HALC ABP the f o l l o w i n g s e r i e s of s t u d i e s were done. B i n d i n g s t u d i e s u s i n g p a r t i a l l y p u r i f i e d h e p a t i c c y t o s o l from c o n t r o l , 4 and 10 day STZ d i a b e t i c r a t s were done. R e s u l t s of these exper iments may be seen in t a b l e VIII and f i g u r e s 3 a - c , and 4. No s i g n i f i c a n t d i f f e r e n c e s in Kd va lues were seen . However, there was a s i g n i f i c a n t decrease in the b i n d i n g c a p a c i t y between e i t h e r u n t r e a t e d c o n t r o l or c i t r a t e d t r e a t e d c o n t r o l r a t s and 4 or 10 day STZ d i a b e t i c s . 49 F i g u r e 3 SATURATION CURVES FOR BINDING STUDIES OF HEPATIC CYTOSOLIC HIGH AFFINITY-LOW CAPACITY FROM CONTROL, 4 AND 10 DAY STREPTOZOTOCIN DIABETIC ADULT MALE RATS A d u l t (240-390 g) male r a t h e p a t i c c y t o s o l , f r a c t i o n a t e d wi th 50 % ammonium s u l f a t e ( 0 . 5 - 1 . 0 mg/mL) from (a) c o n t r o l , (b) 4 and (c) 10 day STZ d i a b e t i c r a t s was prepared as d e s c r i b e d in the Methods s e c t i o n and was incuba ted with v a r y i n g c o n c e n t r a t i o n s of t r i t i a t e d R1881 (0 .05-5 nM), with or w i thout 100 f o l d molar excess R1881, and with 100 f o l d molar excess t r i a m c i n o l o n e ace ton ide f o r 3 hours at 4°C. 5 0 F I G U R E 3 a 1 0 0 BO W E \ 6 0 -0 1 2 3 4 5 6 F r e e (nmol/L) FIGURE 3 b 80-0 1 2 3 4 5 F r e e (nmol/L) 5 2 FIGURE 3 c 8 0 H 53 F i g u r e 4 SCATCHARD PLOT FOR BINDING STUDIES OF THE HEPATIC CYTOSOLIC HIGH AFFINITY-LOW CAPACITY ANDROGEN BINDING PROTEIN IN CONTROL, 4 AND 10 DAY STREPTOZOTOCIN DIABETIC ADULT MALE RATS Data p resented in a s a t u r a t i o n p l o t in f i g u r e 3 was t rans formed and i s p resented in a Sca t chard p l o t . B i n d i n g c a p a c i t y and Kd va lues were determined by l i n e a r r e g r e s s i o n a n a l y s i s of d a t a . F I G U R E 4 S p e c i f i c Bound (fmol/mg) Table VIII THE EFFECT OF 4 AND 10 DAY STZ DIABETES ON THE BINDING PARAMETERS OF THE HEPATIC CYTOSOLIC HIGH AFFINITY-LOW CAPACITY ANDROGEN BINDING PROTEIN Standard assay c o n d i t i o n s were used with h e p a t i c c y t o s o l from unt rea ted c o n t r o l , c i t r a t e b u f f e r i n j e c t e d , and 4 and 10 day STZ d i a b e t i c a d u l t male r a t s (240-390 g ) . Animal Model Kd (nM) B i n d i n g C a p a c i t y H (fmol/mgT Cont ro l 0.30 + 0.02 20.76 + 1.49 43 C i t r a t e c o n t r o l 0.33 + 0.04 22.51 + 2.48 26 4 Day STZ 0.31 + 0.05 9.71 + 1.22 * 22 10 Day STZ 0.36 + 0.04 8.31 + 1.21 * 23 * S i g n i f i c a n t l y d i f f e r e n t from c o n t r o l (P < 0.05) us ing ANOVA and Newman-Keul's range t e s t Tab le IX THE EFFECT OF 4 AND 10 DAY STREPTOZOTOCIN DIABETES ON THE TEMPERATURE STABILITY OF THE HEPATIC CYTOSOLIC HIGH AFFINITY-LOW CAPACITY ANDROGEN BINDING PROTEIN OF ADULT MALE RATS Standard assay c o n d i t i o n s were used, except tha t the 50 % ammonium s u l f a t e f r a c t i o n a t e d c y t o s o l was p r e i n c u b a t e d at 24°C f o r 0 to 6 hours be fo re the s t a r t of the 3 hour 4°C i n c u b a t i o n . 1. Con t ro l versus 4 day STZ d i a b e t i c Time (hours) Kd (nM) B i n d i n g C a p a c i t y (fmol /mgT Cont ro l STZ d i a b e t i c (4 day) Cont ro l STZ d i a b e t i c (4 day) 0 0.19 0.14 10.63 10.07 0.5 0.34 0.08 7.10 4.95 1.5 0.17 0.08 1.47 1.68 3.0 NS NS NS NS 6.0 NS NS NS NS NS denotes no Sca tchard p l o t 5 7 TABLE IX 2. Cont ro l versus 10 day STZ d i a b e t i c Time (hours) Kd (nM) B ind ing c a p a c i ty (finol /mgT Cont ro l STZ d i a b e t i c Con t ro l STZ d i a b e t i c (10 day) 0 0.22 0.13 23.40 4.38 0.5 0.24 0.10 19.36 4.21 1.5 0.35 0.53 8.15 3.92 3.0 NS NS NS NS 6.0 NS NS NS NS NS denotes no s c a t c h a r d p l o t 5 8 2. Temperature s t a b i l i t y of the high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n The lower b i n d i n g c a p a c i t y of the HALC ABP seen i n STZ d i a b e t i c h e p a t i c c y t o s o l may s imply be due to decreased s t a b i l i t y of the HALC ABP prepared from the STZ d i a b e t i c a n i m a l . In order to t e s t t h i s p o s s i b i l i t y p a r t i a l l y p u r i f i e d c y t o s o l from c o n t r o l and d i a b e t i c r a t l i v e r was a l l o w e d to s t a n d , at 24°C in a water bath f o r 0, 0 . 5 , 1 .5, 3 and 6 hours be fore the s t a r t of the i n c u b a t i o n to determine the r e l a t i v e s t a b i l i t y of the HALC ABP. t h i s e x p e r i m e n t was done us ing h e p a t i c c y t o s o l from four and ten day d i a b e t i c r a t s . R e s u l t s are in t a b l e s IX, 1 and 2. The Kd showed no change in t h i s e x p e r i m e n t . The b i n d i n g c a p a c i t y d e c r e a s e d as t ime of p r e i n c u b a t i o n at 24°C i n c r e a s e d . There appears to be no d i f f e r e n c e in the ra te of degradat ion between the HALC ABP prepared from c o n t r o l and 4 day STZ d i a b e t i c r a t l i v e r . There i s no apparent change in the b i n d i n g c a p a c i t y of the HALC ABP prepared from the l i v e r of the 10 day STZ d i a b e t i c r a t , u n t i l at 3 hours i t i s no longer d e t e c t a b l e . 3. E f f e c t of the i n d u c t i o n of s t r e p t o z o t o c i n d i a b e t e s on the s p e c i f i c i t y of the high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n In o rder to determine whether STZ d i a b e t e s had an e f f e c t on s p e c i f i c i t y of the HALC ABP b i n d i n g , i n h i b i t i o n s t u d i e s with s t e r o i d a l and n o n - s t e r o i d a l c o m p e t i t o r s in c o n t r o l and STZ d i a b e t i c s were under taken . Data was p l o t t e d as percent t o t a l bound remain ing versus f r e e c o m p e t i t o r added and as pe rcen t s p e c i f i c bound remain ing versus f r e e c o m p e t i t o r added. I C g n va lues were determined from a l o g i t (pe rcen t s p e c i f i c bound remain ing) versus l og ( f r e e ) p l o t , and by u s i n g the l e a s t squares method of l i n e a r r e g r e s s i o n . Tab le X, 1 and 2, and f i g u r e 5, a and b summarize the r e s u l t s of t h e s e e x p e r i m e n t s . The o r d e r of a f f i n i t y of c o m p e t i t o r s was R1881 = 5a-DHT > proges terone > t e s t o s t e r o n e enanthate = e s t r a d i o l > a n d r o s t e n e d i o n e . 5p-DHT and d i e t h y l s t i 1 b e s t e r o l produced i n s i g n i f i c a n t d i sp lacement of R1881 from s p e c i f i c b i n d i n g s i t e s . R e s u l t s were g e n e r a l l y poorer ( l a r g e s tandard e r r o r s ) i n d i a b e t i c s but showed a s i m i l a r p a t t e r n of af f i ni t y . V I I . E f f e c t of hormone rep lacement on the h e p a t i c c y t o s o l i c high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n 1. E f f e c t s of once d a i l y a d m i n i n s t r a t i o n of protamine z i n c i n s u l i n and twice d a i l y a d m i n i s t r a t i o n of Toronto i n s u l i n O r t i z - C a r a et al (1984) showed r e s t o r a t i o n of the p a t t e r n of s e c r e t i o n and amount of t h y r o t r o p i n s t i m u l a t i n g 6 0 Tab le X THE EFFECT OF 10 DAY STREPTOZOTOCIN DIABETES ON THE SPECIFICITY OF THE HEPATIC CYTOSOLIC HIGH AFFINITY-LOW CAPACITY ANDROGEN BINDING PROTEIN OF MALE RATS 1. Con t ro l r a t s A d u l t (240-390 g) male Wis ta r r a t h e p a t i c c y t o s o l ( 0 . 5 - 1 . ! mg prote in/mL) was prepared and i n c u b a t i o n s conducted as d e s c r i b e d in the Methods s e c t i o n . I C 5 Q va lues were determined us ing a l o g i t - l o g p l o t ana the l e a s t squares method of l i n e a r r e g r e s s i o n . Exper iments were conducted s e v e r a l t i m e s , t h e r e f o r e data i s expressed as average + SEM. Competi t o r s I C 5 Q (nM) N^  Androgens R1881 0.45 + 0.16 a 10 5<x-DHT 1.42 + 0.40 a 6 T e s t o s t e r o n e enanthate 56.60 +_ 15.24 b,c 4 Androstened ione 83.14 + 24.48 c 7 56-DHT * 3 Es t rogens 1 7 6 - E s t r a d i o l 56.7 + 5.44 b,c 5 D i e t h y l s t i 1 b e s t e r o l * 6 P r o g e s t i n s Proges terone 2.40 + 7.88 a,b 7 * a l though some d i sp lacement o c c u r r e d , i t was not c o n s i s t e n t and d id not i n c r e a s e with i n c r e a s i n g c o n c e n t r a t i o n s of compet i to r S i m i l a r l e t t e r s or combinat ions of l e t t e r s i n d i c a t e no s i g n i f i c a n t d i f f e r e n c e at p < 0.05 l e v e l us ing ANOVA and Newman Keuls range t e s t 6 1 Tab le X 2. Ten day STZ d i a b e t i c r a t s A d u l t (240-390 g) male Wis ta r r a t h e p a t i c c y t o s o l ( 0 . 5 - 1 . 5 mg prote in/mL) was prepared and i n c u b a t i o n s conducted as d e s c r i b e d in the Methods s e c t i o n . I C 5 Q va lues were determined us ing a l o g i t - l o g p l o t and the l e a s t squared method of l i n e a r r e g r e s s i o n . Exper iments were conducted s e v e r a l t i m e s , t h e r e f o r e data i s expressed as average + SEM. Competi t o r l i s o ^ Androgens R1881 5a-DHT T e s t o s t e r o n e enanthate 5p-DHT 0.18 + 0.17 a 5 1.66 + 1.28 3 167.25 + 82.18 b 4 * 6 Es t rogens 1 7 p - E s t r a d i o l 111.00 + 66.78 P r o g e s t i ns Proges terone 37.45 + 13.91 * a l though some d i s p l a c e m e n t o c c u r r e d , i t was not c o n s i s t e n t and d id not i n c r e a s e with i n c r e a s i n g c o n c e n t r a t i o n s of competi t o r S i m i l a r l e t t e r s i n d i c a t e no s i g n i f i c a n t d i f f e r e n c e s at the P < 0.05 l e v e l us ing to ANOVA and Newman-Keul's range t e s t No l e t t e r s i n d i c a t e va lues are not s i g n i f i c a n t l y d i f f e r e n t from a o r b 62 F i g u r e 5 SPECIFICITY STUDIES OF THE HIGH AFFINITY-LOW CAPACITY ANDROGEN BINDING PROTEIN IN HEPATIC CYTOSOL FROM CONTROL AND 10 DAY STREPTOZOTOCIN DIABETIC ADULT MALE RATS A d u l t (240-390 g) male Wis ta r r a t h e p a t i c c y t o s o l (0 .5 to 1.5 mg/mL) was incuba ted with 1 nM of t r i t i a t e d R1881 and 100 f o l d molar excess of t r i a m c i n o l o n e a c e t o n i d e in the presence of i n c r e a s i n g c o n c e n t r a t i o n of s e v e r a l s t e r o i d a l and n o n - s t e r o i d a l c o m p e t i t o r s , f o r 3 hours at 4°C. Percentage of s p e c i f i c b i n d i n g was c a l c u l a t e d as the f r a c t i o n of R1881 d i s p l a c e d r e l a t i v e to t o t a l R1881 bound m u l t i p l i e d by 100. Th i s va lue was p l o t t e d as a f u n c t i o n of the l o g a r i t h m of the f r e e c o n c e n t r a t i o n of the c o m p e t i t o r p resent in the i n c u b a t i o n m i x t u r e . F i g u r e a shows t y p i c a l t r i t i a t e d - R 1 8 8 1 c o m p e t i t o r curves f o r R1881, t e s t o s t e r o n e e n a n t h a t e , 56-DHT, a n d r o s t e n e d i o n e , 5a-DHT, e s t r a d i o l , d i e t h y l s t i 1 b e s t e r o l , and progesterone in c o n t r o l a d u l t male W i s t a r r a t s . F i g u r e b shows t y p i c a l t r i t i a t e d R1881 c o m p e t i t o r curves f o r R1881, 5a-DHT, t e s t o s t e r o n e enantha te , 56-DHT, e s t r a d i o l , and progesterone in 10 day STZ d i a b e t i c a d u l t male W i s t a r r a t s . 6 3 FIGURE 5 a 0 - 1 0 1 2 3 4 Log o f Competitor- added i n nM FIGURE 5 b Log Competitor added i n nM Tab le XI THE EFFECT OF TREATMENT WITH REGULAR (TORONTO) AND PROTAMINE ZINC INSULIN ON THE HEPATIC CYTOSOLIC HIGH AFFINITY-LOW CAPACITY ANDROGEN BINDING PROTEIN OF 4 DAY STREPTOZOTOCIN DIABETIC MALE RATS A d u l t (240-390 g) male r a t s were t r e a t e d s . c . with r e g u l a r ( T o r o n t o ) i n s u l i n (15 U/kg, tw ice a day at 0830 and 1700) with PZI (10 U/kg, once a d a y ) . Treatment was s t a r t e d at the time of i n d u c t i o n of d i a b e t e s with STZ. Standard assay c o n d i t i o n s were used . Animal Model Kd (nM) B i n d i n g C a p a c i ty H (fmol/mgT C i t r a t e c o n t r o l 0.33 + 0.04 22.51 + 2.48 26 4 Day STZ 0.31 + 0.05 9.71 + 1.22 * 22 4 Day STZ + PZI 0.60 + 0.19 11.56 + 3.72 * 4 4 Day STZ + Toronto I n s u l i n 0.28 + 0.06 6.66 + 1.30 * 8 * I n d i c a t e s 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 at P < 0.05 l e v e l us ing ANOVA and Newman-Keul's Range t e s t 6 6 hormone, t r i i o d o t h y r o n i n e , and t h y r o x i n e , when 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, in a dose of 15 U/kg, to male Wis ta r r a t s . We attempted to r e s t o r e the HALC ABP us ing the same reg imen. The r e s u l t s of t h i s study are shown i n t a b l e X I . Toronto i n s u l i n a d m i n i s t e r e d twice d a i l y had no r e s t o r a t i v e e f f e c t on the HALC ABP. PZI (10 U/kg) a d m i n i s t r a t i o n once d a i l y a l s o was i n e f f e c t i v e in r e s t o r i n g HALC ABP l e v e l s . Testape® r e s u l t s were n e g a t i v e f o r i n s u l i n t r e a t e d d i a b e t i c s . 2. E f f e c t s of t e s t o s t e r o n e enanthate and t e s t o s t e r o n e enanthate with protamine z i n c i n s u l i n t reatment No change in Kd va lues in e i t h e r t e s t o s t e r o n e enanthate (1 mg/kg) or t e s t o s t e r o n e (1 mg/kg) and PZI (10 U/kg) t r e a t e d 4 day STZ d i a b e t i c r a t s was found (Tab le X I I ) . The decreased b i n d i n g c a p a c i t y was not r e v e r s e d in t e s t o s t e r o n e enanthate t r e a t e d 4 day STZ d i a b e t i c r a t s . Treatment with t e s t o s t e r o n e enanthate and PZI r e s u l t e d in a p a r t i a l r e s t o r a t i o n of the b i n d i n g c a p a c i t y of the HALC ABP to an i n t e r m e d i a t e v a l u e , not s t a t i s t i c a l l y d i f f e r e n t from e i t h e r c o n t r o l or STZ d i a b e t i c s . 3. E f f e c t s of t r i i o d o t h y r o n i n e t reatment In an attempt to r e s t o r e c o n t r o l l e v e l s of the b i n d i n g Tab le XII THE EFFECT OF TREATMENT OF 4 DAY STREPTOZOTOCIN DIABETIC MALE RATS WITH TESTOSTERONE ENANTHATE, OR TESTOSTERONE ENANTHATE WITH PROTAMINE ZINC INSULIN ON THE HEPATIC CYTOSOLIC HIGH AFFINITY-LOW CAPACITY ANDROGEN BINDING PROTEIN A d u l t (240-390 g) male STZ d i a b e t i c r a t s were t r e a t e d subcutaneous ly with t e s t o s t e r o n e enanthate (1 mg/kg subcutaneous ly once d a i l y ) or with t e s t o s t e r o n e enanthate and and protamine z i n c i n s u l i n (10 U/kg, once d a i l y ) . Treatment was s t a r t e d at i n d u c t i o n of d i a b e t e s with STZ. S tandard i n c u b a t i o n c o n d i t i o n s were used . C i t r a t e c o n t r o l 0.33 + 0.04 22.51 + 2.48 a 26 Animal Model Kd (nM) B i n d i n g C a p a c i ty (fmol/mgT N 4 Day STZ 0.31 + 0.05 9.71 + 1.22 b 22 4 Day STZ + 0.41 + 0.07 10.05 + 1.79 b 8 t e s t o s t e r o n e enanthate 4 Day STZ 0.45 + 0.07 15.70 + 2.91 11 + t e s t o s t e r o n e enanthate and ov ine growth hormone S i m i l a r l e t t e r s i n d i c a t e va lues which are not s i g n i f i c a n t l y d i f f e r e n t at the P < 0.05 l e v e l us ing ANOVA and Newman-Keul 1s Range t e s t 6 8 Tab le XIII THE EFFECT OF TREATMENT WITH TRIIODOTHYRONINE ON THE HEPATIC CYTOSOLIC HIGH AFFINITY-LOW CAPACITY ANDROGEN BINDING PROTEIN OF 4 DAY STREPTOZOTOCIN STZ DIABETIC MALE RATS Hepat i c c y t o s o l ( 0 . 5 - 1 . 5 mg prote in/mL) from c o n t r o l and four day STZ and four day STZ d i a b e t i c r a t s t r e a t e d with t r i i o d o t h y r o n i n e a d u l t male r a t s (240-390 g) was assayed a c c o r d i n g to s tandard assay c o n d i t i o n s . Animal Model Kd (nM) B i n d i n g Capaci ty N^  (f mol /m"gT C i t r a t e c o n t r o l 0.33 + 0.04 22.51 + 2.48 26 4 Day STZ 0.31 + 0.05 9.71 + 1.22 * 22 4 Day STZ + 0.12 + 0.01 9.49 + 1.97 * 6 t r i i odothyron ine * I n d i c a t e s s i g n i f i g a n t d i f f e r e n c e s at the P < 0.05 l e v e l us ing ANOVA and Newman-Keul's Range t e s t c a p a c i t y of HALC ABP, t r i i o d o t h y r o n i n e was a d m i n i s t e r e d to STZ d i a b e t i c r a t s . The dose of t r i i o d o t h y r o n i n e used was 3 jig per 100 g d a i l y . Th i s dose has been shown to r e s t o r e normal t h y r o i d index in STZ d i a b e t i c male W i s t a r r a t s ( T a h a l i a n i , 1983) . R e s u l t s of the study may be seen i n t a b l e X I I I ; the hormone t reatment had no e f f e c t on Kd or b i n d i n g c a p a c i t y v a l u e s . 4. E f f e c t s of growth hormone replacment (a) E f f e c t of con t inous i n f u s i o n of ov ine growth hormone by osmotic mini pump Mature male r a t s were imp lan ted wi th minipumps, then STZ was a d m i n i s t e r e d to induce d i a b e t e s . The i n f u s i o n ra te of oGH was 0.02 U/hr over the 4 days of d i a b e t e s . Con t ro l and STZ r a t s were operated on, but no minipumps were i m p l a n t e d . No s i g n i f i c a n t change in Kd va lues occured ( t a b l e X I V ) . The b i n d i n g c a p a c i t y of the HALC ABP in oGH t r e a t e d 4 day STZ d i a b e t i c r a t s was not r e s t o r e d to c o n t r o l v a l u e s . (b) E f f e c t of m u l t i p l e subcutaneous ov ine growth hormone doses R e p l i c a t i o n of the normal male s e c r e t i o n p a t t e r n of oGH was attempted by t r e a t i n g with 7 s . c . doses of oGH ( i n 7 0 Tab le XIV THE EFFECT OF INFUSION OF OVINE GROWTH HORMONE BY MINIPUMP ON THE HEPATIC CYTOSOLIC HIGH AFFINITY-LOW CAPACITY ANDROGEN BINDING PROTEIN OF STREPTOZOTOCIN DIABETIC MALE RATS Ovine growth hormone was a d m i n i s t e r e d in a dose of 0.02 U/hour s t a r t i n g at i n d u c t i o n of d i a b e t e s with STZ in a d u l t (240-390 g) male r a t s . Rats were k i l l e d a f t e r 4 days . C o n t r o l and 4 day STZ d i a b e t i c c o n t r o l r a t s were o p e r a t e d on but no minipump was i m p l a n t e d . Standard assay c o n d i t i o n s were used . Animal Model Kd (nM) B i n d i n g C a p a c i t y H (fmol/mg) C i t r a t e c o n t r o l 0. 33 + 0. 04 22. 51 + 2 .48 a 26 I n t e r n a l c o n t r o l 0. 50 + 0. 07 14. 66 + 2 .60 3 4 Day STZ 0. 55 + 0. 32 7. 66 + 1 .45 3 4 Day STZ + ov ine 0. 35 + 0. 11 7. 16 + 1 .92 b 6 growth hormone S i m i l a r l e t t e r s i n d i c a t e va lues which are not s i g n i f i c a n t l y d i f f e r e n t at the P < 0.05 l e v e l us ing ANOVA and Newman-Keul 1 s Range t e s t Tab le XV THE EFFECT OF MULTIPLE SUBCUTANEOUS DOSES OF OVINE GROWTH HORMONE ON THE HEPATIC CYTOSOLIC HIGH AFFINITY-LOW CAPACITY ANDROGEN BINDING PROTEIN OF FOUR DAY STREPTOZOTOCIN DIABETIC MALE RATS Ovine growth hormone was a d m i n i s t e r e d s . c . 7 t imes a day (0240, 0615, 1015, 1415, 1815, 2100, and 2340) in a dose of 30 ng in s a l i n e , pH 8 . 5 , per r a t per dose s t a r t i n g at i n d u c t i o n of d i a b e t e s with STZ in a d u l t (240-390 g) male r a t s . Cont ro l and STZ d i a b e t i c s were a d m i n i s t e r e d s a l i n e at pH 8.5 i n the same way. Rats were k i l l e d 4 days a f t e r i n d u c t i o n of d i a b e t e s . S t a n d a r d assay c o n d i t i o n s were u s e d . Animal Model Kd (nM) B ind ing C a p a c i t y ^ (fmol/mgT C i t r a t e c o n t r o l 0.33 + 0.04 22.51 + 2.48 a 26 I n t e r n a l Cont ro l 0.20 + 0.01 14.75 + 1.93 4 4 Day STZ 0.31 + 0.11 9.25 + 4.26 4 4 Day STZ + 0.33 + 0.11 ov ine growth hormone 7.28 + 2.88 b 7 S i m i l a r l e t t e r s i n d i c a t e va lues tha t are not s i g n i f i c a n t l y d i f f e r e n t from each other at the P < 0.05 l e v e l us ing ANOVA and Newman-Keul's Range t e s t 7 2 s a l i n e , pH 8 . 5 ) , d a i l y . C o n t r o l and 4 day STZ d i a b e t i c r a t s were g iven 3 d a i l y doses of s a l i n e (pH 8 . 5 ) . R e s u l t s in t a b l e XV show no s i g n i f i c a n t changes in Kd v a l u e s , and no r e s t o r a t i o n of the the b i n d i n g c a p a c i t y of the 4 day STZ d i a b e t i c oGH t r e a t e d group to c o n t r o l l e v e l s . ( c ) E f f e c t of m u l t i p l e dos ing of ov ine growth hormone us ing a vena cava c a t h e t e r Intravenous c a t h e t e r s were implanted i n t o the vena  c a v a . Ovine GH was a d m i n i s t e r e d 7 t imes a day, in a dose of 30 u g/rat/dose in s a l i n e , pH 8.5 in order to more a c c u r a t e l y d u p l i c a t e normal oGH s e c r e t o r y p a t t e r n s in the male r a t . Cont ro l and 4 day STZ r a t s had vena cava c a t h e t e r s implanted through which s a l i n e , pH 8 . 5 , was a d m i n i s t e r e d 7 t imes a day. T e c h n i c a l d i f f i c u l t y encountered in the use of the vena cava c a t h e t e r s reduce the number of an ima ls to the p o i n t tha t s t a t i s t i c a l a n a l y s i s was not p o s s i b l e . S t i l l , from t a b l e XVI i t i s apparent tha t c o n t r o l and 4 day STZ b i n d i n g c a p a c i t y va lues are i d e n t i c a l and c o n s i d e r a b l y below normal c o n t r o l b i n d i n g c a p a c i t y v a l u e s . In t h i s exper iment , vena cava c a t h e t e r s were implanted under ha lothane a n a e s t h e t i c . Halothane has been s u s p e c t e d of c a u s i n g h e p a t o t o x i c i t y (Brown and S i p e s , 1977) , so an exper iment us ing e t h e r , the a n a e s t h e t i c commonly used in our l a b o r a t o r y , was done. Tab le XVI THE EFFECT OF MULTIPLE INTRAVENEOUS DOSES OF OVINE GROWTH HORMONE BY VENA CAVA CATHETER ON THE HEPATIC CYTOSOLIC HIGH AFFINITY-LOW CAPACITY ANDROGEN BINDING PROTEIN OF STREPTOZOTOCIN DIABETIC MALE RATS Ovine growth hormone was a d m i n i s t e r e d in a dose of 30 ug i n s a l i n e , pH 8 . 5 , 7 t imes a day (0240, 0615, 1015, 1415, 1815, 2100, and 2340) by vena cava c a t h e t e r s t a r t i n g at the i n d u c t i o n of d i a b e t e s with STZ i n a d u l t male r a t s (240-390 g ) . Cont ro l and STZ d i a b e t i c c o n t r o l s a l s o had vena cava c a t h e t e r s implanted and s a l i n e , pH 8.5 was a d m i n i s t e r e d 7 t imes a day. Rats were k i l l e d 4 days a f t e r i n d u c t i o n of d i a b e t e s . S tandard assay c o n d i t i o n s were used . Animal Model Kd (nM) B i n d i n g C a p a c i t y N[ (fmol / m g T C i t r a t e c o n t r o l 0.33 + 0.04 22.51 + 2.48 26 I n t e r n a l c o n t r o l 0.08 + 0.02 8.91 + 2.02 3 4 Day STZ 0.11 + 0.03 8.94 + 0.17 2 4 Day STZ + ov ine 0.15 + 0.03 6.17 + 1.16 3 growth hormone S t a t i s t i c a l t e s t s were not done due to the low number of an imals used 7 4 ( d ) E f f e c t s of e ther versus ha lothane general a n a e s t h e s i a in c o n t r o l r a t s Vena cava c a t h e t e r s were implanted i n t o c o n t r o l r a t s under e t h e r or h a l o t h a n e a n a e s t h e t i c . An ima l s were k i l l e d 4 days l a t e r ; c a n n u l a s were not u s e d . A l t h o u g h per formed on a l i m i t e d number of a n i m a l s , r e s u l t s ( t a b l e XVII) show tha t there i s no advantage to e i t h e r e ther or ha lothane as a general a n a e s t h e t i c f o r our purposes . ( e ) E f f e c t s of m u l t i p l e t a i l ve in i n j e c t i o n s of ov ine growth hormone P u r s u i n g r e s t o r a t i o n of the normal oGH s e c r e t o r y p a t t e r n , we next d id exper iments in which oGH (30 ng/dose in s a l i n e , pH 8.5) was a d m i n i s t e r e d 4 t imes a day us ing the t a i l v e i n . Again c o n t r o l and 4 day STZ c o n t r o l s r e c e i v e d equal amounts of s a l i n e , pH 8 . 5 . R e s u l t s are g iven in t a b l e X V I I I . No s i g n i f i c a n t d i f f e r e n c e in Kd v a l u e s , no r e s t o r a t i o n to c o n t r o l b i n d i n g c a p a c i t i e s occured in oGH t r e a t e d 4 day STZ d i a b e t i c r a t s . Tab le XVII THE EFFECT OF HALOTHANE VERSUS ETHER ANAESTHESIA ON THE HEPATIC CYTOSOLIC HIGH AFFINITY-LOW CAPACITY ANDROGEN BINDING PROTEIN OF ADULT MALE RATS Vena cava c a t h e t e r s were imp lanted i n t o a d u l t (240-390 g) male r a t s ) under e ther or ha lothane a n a e s t h e s i a . C a t h e t e r s were not used . Rats were k i l l e d four days a f t e r s u r g e r y . S tandard assay c o n d i t i o n s were used . Animal Model Kd (nM) B i n d i n g Capaci ty ( f mo mg) Cont ro l 0.30 + 0.02 20.76 + 1.49 (43) E ther 0.26 13.81 E ther 0.19 12.06 Halothane 0.26 14.59 Halothane 0.48 34.00 ( ) i n d i c a t e s N S t a t i s t i c a l a n a l y s i s was not done due to the low number of an imals s t u d i e d . Tab le XVIII THE EFFECT OF MULTIPLE INTRAVENEOUS DOSES OF OVINE GROWTH HORMONE BY THE TAIL VEIN ON THE HEPATIC CYTOSOLIC HIGH AFFINITY-LOW CAPACITY ANDROGEN BINIDNG PROTEIN OF 4 DAY STREPTOZOTOCIN DIABETIC MALE RATS Ovine growth hormone in a dose of 30 ng in s a l i n e , pH 8 . 5 , was a d m i n i s t e r e d by the t a i l ve in 4 t imes a day (0900, 1300, 1700, 2100 hours) s t a r t i n g with the i n d u c t i o n of d i a b e t e s with STZ in a d u l t (240-390 g) male r a t s . Con t ro l and STZ c o n t r o l s were i n j e c t e d with s a l i n e , pH 8.5 in the same way. Rats were k i l l e d 4 days a f t e r i n d u c t i o n of d i a b e t e s . S tandard assay c o n d i t i o n s were used . Animal Model Kd (nM) Bi ndi ng Capaci ty ft (fmol / m g T C i t r a t e c o n t r o l 0.33 + 0.04 22.51 + 2.48 26 I n t e r n a l c o n t r o l 0.25 + 0.03 20.98 + 3.20 8 4 Day STZ 0.33 + 0.06 7.75 + 0.75 * 6 4 Day STZ 0.44 + 0.18 5.41 + 1.93 * 4 + ovine growth hormone * I n d i c a t e s s i g n i f i c a n t d i f f e r e n c e from e i t h e r c o n t r o l at the P < 0.05 l e v e l us ing ANOVA and Newman-Keul 1s Range t e s t ( f ) E f f e c t s of m u l t i p l e t a i l ve in i n j e c t i o n s of ov ine growth hormone, d a i l y i n j e c t i o n s of t e s t o s t e r o n e enantha te , s u b c u t a n e o u s l y , and d a i l y i n j e c t i o n of protamine z i n c i n s u l i n , subcutaneous ly P r e v i o u s r e s u l t s us ing PZI and t e s t o s t e r o n e enanthate rep lacement showed p a r t i a l r e s t o r a t i o n of HALC ABP l e v e l s . T h e r e f o r e , to determine whether oGH, t e s t o s t e r o n e e n a n t h a t e , and PZI were a l l r e q u i r e d f o r normal HALC ABP b i n d i n g c a p a c i t y l e v e l s , a set of exper iments in which d a i l y subcutaneous ly PZI (10 U/kg) and t e s t o s t e r o n e enanthate (lmg/kg) dos ing a long with 4 d a i l y t a i l ve in doses of oGH (30 ug) was c a r r i e d ou t . Cont ro l and 4 day STZ r a t s r e c e i v e d s a l i n e , pH 8 . 5 , 4 t imes a day and d a i l y s . c . i n j e c t i o n s of corn o i l , which was the v e h i c l e used to d e l i v e r t e s t o s t e r o n e enantha te . R e s u l t s ( t a b l e XIX) show no s i g n i f i c a n t change in Kd v a l u e s , and no r e s t o r a t i o n to c o n t r o l b i n d i n g c a p a c i t i e s of 4 day STZ d i a b e t i c t e s t o s t e r o n e enanthate , PZI , and oGH t r e a t e d a n i m a l s . 7 8 Tab le XIX THE EFFECT OF SUBCUTANEOUS ADMINISTRATION OF TESTOSTERONE ENANTHATE AND PROTAMINE ZINC INSULIN WITH MULTIPLE TAIL VEIN INJECTIONS OF OVINE GROWTH HORMONE ON THE HEPATIC CYTOSOLIC HIGH AFFINITY-LOW CAPACITY ANDROGEN BINDING PROTEIN OF 4 DAY STREPTOZOTOXIN DIABETIC MALE RATS A d u l t (240-390 g) male r a t s were t r e a t e d once d a i l y w i th TE , 1 mg/kg s . c ; with PZI , 10 U/kg, s . c ; and with oGH four t imes a day (0900, 1300, 1700, 2100 hours) in a dose of 30 ng in s a l i n e , pH 8.5 i n j e c t e d i n t o the t a i l v e i n , s t a r t i n g at the i n d u c t i o n of d i a b e t e s with STZ. Con t ro l and STZ c o n t r o l s r e c e i v e d equal amounts of corn o i l s . c . once a day, and s a l i n e , pH 8 . 5 , i n t o the t a i l ve in 4 t imes a day. Rats were k i l l e d 4 days a f t e r the i n d u c t i o n of d i a b e t e s . S tandard assay c o n d i t i o n s were used . Animal Model Kd (nM) B i n d i n g C a p a c i t y (fmol/mg) N C i t r a t e c o n t r o l 0. 33 + 0. 04 22.51 + 2.48 26 I n t e r n a l c o n t r o l 0. 23 + 0. 03 24.92 + 2.88 6 4 Day STZ 0. 23 + 0. 04 7.82 + 1.19 * 4 4 Day STZ t r e a t e d 0. 15 + 0. 06 10.12 + 1.02 * 4 with 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 , and ov ine growth hormone * I n d i c a t e s 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 va lue at P < 0.05 l e v e l us ing ANOVA and Newman-Keul's Range t e s t DISCUSSION I . Ammonium s u l f a t e f r a c t i o n a t i o n Ammonium s u l f a t e f r a c t i o n a t i o n was used to p a r t i a l l y p u r i f y the HALC ABP in t h i s study ( t a b l e I ) . We found tha t 0-50 % f r a c t i o n a t i o n produced b i n d i n g with a Kd c h a r a c t e r i s t i c of the HALC ABP seen in whole c y t o s o l , and gave a 2 to 3 f o l d p u r i f i c a t i o n of the HALC ABP based on i n c r e a s e in b i n d i n g c a p a c i t y ( t a b l e I , and I I I ) . The amount of ammonium s u l f a t e r e q u i r e d to f r a c t i o n a t e our h e p a t i c HALC ABP i s d i f f e r e n t from the amount of ammonium s u l f a t e used by o thers to p a r t i a l l y p u r i f y the h e p a t i c ABP, and the p r o s t a t i c AR. E a r l y work done with ammonium s u l f a t e f r a c t i o n a t i o n of h e p a t i c c y t o s o l by Ota e_t aJU 1981, r e s u l t e d in the sed imenta t ion of an ABP in the 30 % ammonium s u l f a t e p e l l e t of h e p a t i c c y t o s o l . P r e c i p i t a t i o n was performed with 40 and 50 % as wel l but dpm/mg p r o t e i n was maximum at 30 %. The sed imenta t ion c o e f f i c i e n t of t h i s p r o t e i n was not de te rmined . A l though s p e c i f i c i t y s t u d i e s of an ABP of whole c y t o s o l had been done by t h i s group, no s t u d i e s on the ammonium s u l f a t e p r e c i p i t a t e d ABP were r e p o r t e d in t h i s or subsequent p a p e r s . Th i s may be important because we found an a t y p i c a l Kd f o r the ABP in the 0-33 % f r a c t i o n a t i o n . Th i s may i n d i c a t e the presence of more than 1 or a f a m i l y of AR, as Lea e t a l , 8 0 1979, suggested e x i s t in the p r o s t a t e . Rumbaugh e t al , 1984, used ammonium s u l f a t e p r e c i p i t a t i o n to decrease n o n - s p e c i f i c b i n d i n g because i t was i m p o s s i b l e to ob ta in a 3 S c a t c h a r d p l o t us ing [ H] 5a-DHT in whole c y t o s o l . T h e r e f o r e , comparison of change due to the p r e c i p i t a t i o n procedure i s i m p o s s i b l e . Decker and L e v i n s o n , 1985, used ammonium s u l f a t e p u r i f i c a t i o n , and they , t o o , found an 8S androgen s p e c i f i c b i n d i n g p r o t e i n in the 0-33 % f r a c t i o n , and no s p e c i f i c b i n d i n g in the 33-60 % f r a c t i o n . No c h a r a c t e r i z a t i o n of the ABP found in the 0-33 % f r a c t i o n was done. Ammonium s u l f a t e p u r i f i c a t i o n of p r o s t a t i c c y t o s o l a l s o r e s u l t s in s e p a r a t i o n of 2 AR 1 s with d i f f e r e n t s t e r o i d s p e c i f i c i t i e s . Fang and L i a o , 1971, and Verhoeven et al , 1975, found an AR of h igher a f f i n i t y p r e c i p i t a t e d in 35 % ammonium s u l f a t e , and a second c l a s s of AR p r e c i p i t a t e d in the 50-60 % ammonium s u l f a t e f r a c t i o n . Mainwaring and P e t e r k e n , 1971, found 2 AR 1 s in f r a c t i o n a t e d p r o s t a t i c c y t o s o l , the 8 S form p r e c i p a t e d at 33 % ammonium s u l f a t e , and the 3.5 S form d id not c o - p r e c i p i t a t e . Because of the d i f f e r e n c e s in amount of ammonium s u l f a t e r e q u i r e d to f r a c t i o n a t e the h e p a t i c versus p r o s t a t i c AR, a sucrose d e n s i t y g r a d i e n t study to determine sed imenta t ion c o e f f i c i e n t of our HALC ABP would be b e n i f i c i a l in t e s t i n g our b e l i e f that the f r a c t i o n a t e d HALC ABP i s the same as the HALC ABP from whole c y t o s o l . 8 1 Freeze s torage (-80°C) of the p e l l e t prepared us ing 50 % ammonium s u l f a t e ( f i g u r e 2) proved s u c c e s s f u l f o r time p e r i o d s of up to 60 days , and a p p a r e n t l y d id not change the AR. S i m i l a r p e l l e t s prepared from p r o s t a t e c y t o s o l and s t o r e d f rozen f o r over 1 month had a changed i n i t i a l v e l o c i t y of i n t e r a c t i o n with 5a-DHT a c c o r d i n g to F e i t and Muldoon ( 1 9 8 3 ) . A more r i g o r o u s study t o examine changes in the HALC ABP with time i s warranted in l i g h t of the r e s u l t s of Fe i t an d Muldoon. I I . I n c l u s i o n of t r i a m c i n o l o n e ace ton ide in the h e p a t i c c y t o s o l i c high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n assay T r i a m c i n o l o n e ace ton ide i s used to block b i n d i n g of R1881 to a second, lower a f f i n i t y - h i g h e r c a p a c t i y b i n d i n g p r o t e i n p r e s e n t in the h e p a t i c c y t o s o l (Eagon et al , 1983, E i s e n f e l d et al , 1983, Kyakumoto e t a l , 1984, and Sunahara et a l , 1985). The reason f o r the suggest ion tha t the lower a f f i n i t y ABP to which R1881 binds in h e p a t i c c y t o s o l i s the g l u c o c o r t i c o i d r e c e p t o r i s as f o l l o w s . R1881 b inds to the c y t o s o l i c p r o g e s t i n r e c e p t o r in the p r o s t a t e of r a t and humans (Zava et a l , 1979, and Menon e t a l , 1978), and some b e l i e v e R1881 binds to a p r o g e s t i n r e c e p t o r in r a t l i v e r c y t o s o l (Lev inson and Decker , 1985). However, Yamada 8 2 and M i y a j i , 1982, and Kyakumoto et a l , 1984, found tha t a c y t o s o l i c p r o g e s t i n r e c e p t o r i s p resent in minimal amounts, i f any, in male l i v e r c y t o s o l . Th i s appears to be the case i n human l i v e r c y t o s o l , too (Aten et a l , 1983). The g l u c o c o r t i c o i d r e c e p t o r i s p resent in the 8S peak of g l y c e r o l d e n s i t y g r a d i e n t c e n t r i f u g a t i o n a n a l y s i s of male r a t h e p a t i c c y t o s o l (Kyakumoto et a l , 1984), and i t i s most l i k e l y the lower a f f i n i t y - h i g h e r c a p a c i t y b i n d i n g p r o t e i n to which R1881 b inds i n the absence of t r i a m c i n o l o n e a c e t o n i d e (Kyakumoto et a l , 1984, and Sunahara et al , 1985). P a r t i a l p u r i f i c a t i o n with ammonium s u l f a t e was s u c c e s s f u l in s e p a r a t i n g much of the n o n - s p e c i f i c b i n d i n g from the HALC ABP. I t was p o s s i b l e tha t p a r t i a l p u r i f i c a t i o n a l s o separa ted the low a f f i n i t y ( g l u c o c o r t i c o i d r e c e p t o r ) ABP of the h e p a t i c c y t o s o l from the high a f f i n i t y (HALC) ABP. Exper iments were done in which t r i a m c i n o l o n e a c e t o n i d e was not i n c l u d e d in the i n c u b a t i o n mixture ( f i g u r e s 1, a and b ) . Two or more b i n d i n g s i t e s were e v i d e n t i n s a t u r a t i o n b i n d i n g curves and S c a t c h a r d p l o t s . T h e r e f o r e i n c l u s i o n of t r i a m c i n o l o n e a c e t o n i d e , 100 f o l d molar e x c e s s , was c o n t i n u e d . I I I . A r t i f a c t u a l r e s u l t s from blood contamina t ion A r e p o r t which showed serum n o n - s p e c i f i c b i n d i n g 8 3 contaminants l e d to i n a c c u r a c i e s in l i v e r p o l y c y c l i c hydrocarbon b i n d i n g assays ( P o e l l i n g e r et a l , 1983) suggested the p o s s i b i l i t y of another confound ing b i n d i n g p r o t e i n be ing present in the p a r t i a l l y p u r i f i e d h e p a t i c c y t o s o l . Th i s r e p o r t a l s o suggested tha t adequate p e r f u s a t e volume cou ld minimize or e l i m i n a t e these con taminants . We found (Tab le I I I ) no d i f f e r e n c e s in Kd or in b i n d i n g c a p a c i t y i n b i n d i n g s t u d i e s done w i th whole and f r a c t i o n a t e d c y t o s o l from l i v e r s pe r fused with 10 or 100 mL TEDM b u f f e r One 50 % ammonium s u l f a t e f r a c t i o n a t e d c y t s o l i c sample from a l i v e r per fused with 100 mL TEDM b u f f e r had a b i n d i n g c a p a c i t y much lower than e x p e c t e d , however, t h i s sample a l s o had a markedly d i f f e r e n t Kd v a l u e . The reason f o r t h i s d i f f e r e n c e may have been p h y s i c a l trauma r e s u l t i n g from p e r f u s i o n of the l i v e r with 100 mL of b u f f e r . IV. Kd and b i n d i n g c a p a c i t y va lues of the h e p a t i c high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n S c a t c h a r d a n a l y s i s of the HALC ABP of mature male Wis ta r r a t s ( t a b l e s IV, V, and VIII) r e s u l t e d in a Kd of 0.30 + 0.02 nM and a b i n d i n g c a p a c i t y of 20.8 + 1.5 fmol/mg p r o t e i n (N = 4 3 ) . Others have r e p o r t e d the f o l l o w i n g v a u l e s , Kd ( i n nM): 25 (Kyakumoto e t a l , 1 9 8 4 ) , 2.3 (Lev inson and Decker , 1985), 0.3 (Sunahara et a l , 1985), 1.5 (Eagon et a l , 1984), and 0.10 (Turocy et al , 8 4 1985) . B i n d i n g c a p a c i t y va lues ( i n fmol/mg p r o t e i n ) a r e : 26.3 (Kyakumoto et a l , 1984), 18.8 (Lev inson and Decker , 1985) , 5.9 (Sunahara et al , 1985), 10-15 (Eagon et al , 1983 a and b, and 1984), 15 (Turocy et a l , 1985) . A l l va lues r e s u l t e d from the use of R1881 i n b i n d i n g s t u d i e s . B a n i s t e r et a l , 1985b, r e p o r t e d va lues us ing m i b o l e r o n e , a n o t h e r s y n t h e t i c a n d r o g e n . They found a Kd o f 0 .86 +_ 0.4 nM, and a b i n d i n g c a p a c i t y of 8.36 _+ 2.77 fmol/mg. It i s u n l i k e l y tha t d i f f e r e n c e s seen are caused by s t r a i n d i f f e r e n c e s , s i n c e a l l except Lev inson and Decker used W i s t a r r a t s . However, d i f f e r e n c e s in pH, in whole versus ammonium s u l f a t e p a r t i a l l y p u r i f i e d c y t o s o l , i n Sca t chard a n a l y s i s versus sucrose d e n s i t y a n a l y s i s , or in t i s s u e : b u f f e r homogen iza t ion volume r a t i o s may c o n t r i b u t e to d i f f e r e n c e s seen in Kd and b i n d i n g c a p a c i t y v a l u e s . I t shou ld be noted tha t a l l the r e p o r t e d va lues are remarkably c l o s e , c o n s i d e r i n g the d i f f e r e n c e s in techn iques used . V. S p e c i f i c i t y of the h e p a t i c high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n F u r t h e r c h a r a c t e r i z a t i o n of the h e p a t i c HALC ABP by d e t e r m i n a t i o n of s p e c i f i c i t y of b i n d i n g has been done by s e v e r a l groups . Compet i tor s t u d i e s done by Lev inson and Decker (1985) , were performed in the absence of t r i a m c i n o l o n e a c e t o n i d e . Th i s group used sucrose d e n s i t y 8 5 g r a d i e n t s to ana lyze r e s u l t s of b i n d i n g s t u d i e s . However, the g l u c o c o r t i c o i d r e c e p t o r to which R1881 a l s o b inds i n the absence of t r i a m c i n o l o n e a c e t o n i d e , i s found in the 8S f r a c t i o n , as i s the AR (Kyakumoto et a l , 1984). As a r e s u l t , Lev inson and Decker ' s compet i to r s t u d i e s i n v o l v e d the d i sp lacement of R1881 from both g l u c o c o r t i c o i d r e c e p t o r and AR. Eagon et al , ( 1983 a and b ) , r e p o r t e d tha t only R1881 and 5a-DHT compete f o r the AR s i t e ; however a complete r e p o r t of these data i s not y e t a v a i l a b l e . Kyakumoto e_t a l , 1984, and Sunahara et a l , 1985, d i d compet i to r s t u d i e s with and wi thout t r i a m c i n o l o n e . In i n c u b a t i o n s c o n t a i n i n g t r i a m c i n o l o n e a c e t o n i d e , Kyakumoto et a l , 1984, found the order of a b i l i t y to d i s p l a c e 10 nM [ 3 H]R1881 from the r e c e p t o r wi th 100 f o l d excess c o m p e t i t o r was: R1881 > t e s t o s t e r o n e = androstened ione > 5a-DHT. E s t r a d i o l , C o r t i s o l , t r i a m c i n o l o n e ace ton ide and proges terone had no a b i l i t y to d i s p l a c e [ 3 H]R1881. Sunahara et a l , 1985, found g e n e r a l l y s i m i l a r r e s u l t s . The order of a b i l i t y to d i s p l a c e 1 nM [ 3 H]R1881 from the r e c e p t o r with 100 f o l d e xce ss of c o m p e t i t o r was as f o l l o w s : 5a-DHT > t e s t o s t e r o n e > R1881 > andros tend ione > e s t r a d i o l . D i e t h y l s t i 1 b e s t e r o l , p r o g e s t e r o n e , C o r t i s o l , and t r i a m c i n o l o n e a c e t o n i d e had no d i s p l a c i n g a b i l i t y . A l though the order of d i s p l a c i n g a b i l i t y d i f f e r s , R1881, t e s t o s t e r o n e and andros tened ione were c l o s e l y c l u s t e r e d in both s t u d i e s . Kyakumoto and Sunahara found some d i f f e r e n c e s in the a b i l i t y of 5a-DHT to 8 6 3 d i s p l a c e [ H]R1881. In the p resent study the order of d i sp lacement of 1 nM [ 3 H]R1881 with 100 f o l d excess c o m p e t i t o r i s : R1881 = 5a-DHT > proges terone > t e s t o s t e r o n e enanthate = andros tend ione = e s t r a d i o l , and no d i sp lacement was seen with d i e t h y l s t i 1 b e s t e r o l . In these th ree s t u d i e s there was agreement in the high s p e c i f i c i t y of the AR f o r R1881 and 5a-DHT; however in our s t u d i e s the HALC ABP had g r e a t e r a f f i n i t y f o r e s t r a d i o l and p r o g e s t e r o n e and l e s s f o r a n d r o s t e n e d i o n e . The reason f o r t h i s d i s c r e p a n c y i s u n c l e a r . The most obvious c o n c l u s i o n was tha t c o - p u r i f i c a t i o n of the g l u c o c o r t i c o i d r e c e p t o r o c c u r e d . The amount of t r i a m c i n o l o n e ace ton ide i n c l u d e d in the i n c u b a t i o n may have been inadequate to mask a l l of the g l u c o c o r t i c o i d r e c e p t o r . We saw no ev idence f o r t h i s in S c a t c h a r d p l o t s ( f i g u r e l b ) , where n o n - l i n e a r i t y of the p l o t i s i n d i c a t i v e of more than 1 b i n d i n g s i t e . I n c r e a s i n g amounts of t r i a m c i n o l o n e a c e t o n i d e d i d not s i g n i f i c a n t l y reduce the b i n d i n g c a p a c i t y of the HALC ABP in male h e p a t i c 50 % ammonium s u l f a t e f r a c t i o n a t e d c y t o s o l ( t a b l e I V ) . Lea et a l , 1979, has suggested a f a m i l y of AR in the p r o s t a t e ; perhaps we s e l e c t i v e l y p r e c i p i t a t e d a subtype of h e p a t i c AR which had h igher p r o g e s t i n a f f i n i t y . 8 7 V I . Sex and age d i f f e r e n c e s i n the h e p a t i c high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n Sex and age d i f f e r e n c e s in the HALC ABP were i n v e s t i g a t e d us ing 50 % ammonium s u l f a t e f r a c t i o n a t e d c y t o s o l . P rev ious exper iments to determine sex s p e c i f i c i t y have produced i n c o n s i s t e n t r e p o r t s between l a b o r a t o r i e s . P a r t i a l p u r i f i c a t i o n with 50 % ammonium s u l f a t e enab les q u a n t i t a t i o n of s m a l l e r amounts of HALC ABP and should more a c c u r a t e l y answer the q u e s t i o n of the presence of small amounts of HALC ABP. In our study immature male and female r a t s had equal amounts of HALC ABP which were s i m i l a r to those found in mature f e m a l e s , and which was 1/4 to 1/2 the amount found in mature males . Others have r e p o r t e d no HALC ABP in immature r a t s (Eagon e t al , 1984), low amounts i n immature males , and none in immature females (Sunahara et a l , 1985). The androgen b i n d i n g s i t e s t u d i e d by Rumbaugh and L u c i e r , 1982, and Rumbaugh et a l , 1984, was absent in immature r a t s . However we q u e s t i o n whether t h i s 4S p r o t e i n i s the same as the high a f f i n i t y b i n d i n g p r o t e i n s t u d i e d by o t h e r s , because of the 4S s e d i m e n t a t i o n c o e f f i c i e n t , unknown s p e c i f i c i t y and the a b i l i t y to measure t h i s b i n d i n g p r o t e i n wi thout the s t a b i l i z i n g e f f e c t of molybdate . In our s tudy , mature female W i s t a r ' r a t s appear to have 1/3 the amount of h e p a t i c HALC ABP tha t i s found in mature male r a t s ( t a b l e I V ) . Others r e p o r t h e p a t i c AR present in 8 8 equa l amounts in mature male and female r a t s (Eagon e t a l , 1983a and b, and Turocy et a l , 1985), or absent in mature females (Lev inson and Decker , 1985, and Sunahara e t a l , 1985) . I n c r e a s i n g amounts of t r i a m c i n o l o n e ace ton ide ( t a b l e IV) r e s u l t e d in an apparent r e d u c t i o n in the amount of HALC ABP in females but not in ma les . The change in Kd of the HALC ABP in male with 100 versus 500 f o l d excess t r i a m c i n o l o n e aceton ide may i n d i c a t e compet i t i on of t r i a m c i n o l o n e f o r the HALC ABP, a l though t h i s has not been demonstrated in compet i to r s t u d i e s done with t r i a m c i n o l o n e in whole c y t o s o l . Because t r i a m c i n o l o n e ace ton ide and R1881 are known to b ind to p r o g e s t i n r e c e p t o r s in the r a t p r o s t a t e (Zava e t a l , 1979), they may have been b i n d i n g to an h e p a t i c p r o g e s t i n r e c e p t o r not p resent in males (Kyakumoto e t a l , 1984) . The presence of a p r o g e s t i n r e c e p t o r in female r a t l i v e r c y t o s o l has not been i n v e s t i g a t e d . It i s u n l i k e l y tha t the t r i a m c i n o l o n e aceton ide r e d u c i b l e b i n d i n g i s due to an i n c r e a s e d amount of g l u c o c o r t i c o i d r e c e p t o r , which i s a l s o seen in males (Kyakumoto e t a l , 1984). The presence of a p r o g e s t i n r e c e p t o r in female r a t l i v e r c y t o s o l has not been i n v e s t i g a t e d . It i s u n l i k e l y tha t the t r i a m c i n o l o n e aceton ide r e d u c i b l e b i n d i n g i s due to an i n c r e a s e d amount of g l u c o c o r t i c o i d r e c e p t o r , which i s a l s o seen in males , because the Kd of g l u c o c o r t i c o i d r e c e p t o r i s c l e a r l y much l e s s than the ABP ( f i g u r e 1, a and b) and the 8 9 measured Kd of the h e p a t i c HALC ABP of females ( t a b l e s IV and V) i s c l o s e r to the Kd of the HALC ABP of males than the Kd of the g l u c o c o r t i c o i d r e c e p t o r . The HALC ABP of female r a t s r e q u i r e s f u r t h e r i n v e s t i g a t i o n . R e s u l t s of c a s t r a t i o n of mature male r a t s are in t a b l e V I . There i s no change in the Kd of b i n d i n g c a p a c i t y a f t e r one week of c a s t r a t i o n . Sunahara e t a l , 1985, showed t h a t the Kd and b i n d i n g c a p a c i t y of the HALC ABP d id not change at 18 hours , 4 or 10 days a f t e r c a s t r a t i o n . Eagon et al , 1984, found a temporary enhancement of the c y t o s o l i c AR at 48 hours a f t e r c a s t r a t i o n , then a decrease to u n d e t e c t a b l e l e v e l s 30 days a f t e r c a s t r a t i o n . R e s u l t s appear to be c o n s i s t e n t between groups , however v e r i f i c a t i o n of the i n c r e a s e at 48 hours , and the absence of the HALC ABP 30 days a f t e r c a s t r a t i o n i s n e c e s s a r y . The b i n d i n g c a p a c i t y of n e o n a t a l l y c a s t r a t e d male r a t s was low or u n d e t e c t a b l e . N e o n a t a l l y c a s t r a t e d t e s t o s t e r o n e - t r e a t e d r a t s a l s o had low b i n d i n g c a p a c i t i e s , but u n l i k e n e o n a t a l l y c a s t r a t e d r a t s , produced r e l i a b l e S c a t c h a r d p l o t s . Severa l p o s s i b i l i t i e s f o r these r e s u l t s e x i s t . S t r e s s dur ing the neonata l p e r i o d may have i r r e v e r s i b l e e f f e c t s on the a b i l i t y to produce normal l e v e l s of HALC ABP at m a t u r i t y . I m p r i n t i n g , where normal a d u l t l e v e l s of enzyme a c t i v i t y o r b i n d i n g p r o t e i n content are dependent on in u tero or neonata l surges of a hormone, may occur with the HALC ABP. Th i s does occurs with o ther h e p a t i c s t e r o i d b i n d i n g prote ins . (S1 oop et a 1 , 9 0 1983). To t e s t t h i s p o s s i b i l i t y , a d m i n i s t r a t i o n of t e s t o s t e r o n e to mature n e o n a t a l l y c a s t r a t e d and n e o n a t a l l y c a s t r a t e d , p r e p u b e r t a l l y , a n d r o g e n - i m p r i n t e d r a t s would be n e c e s s a r y . V I I . E f f e c t s of d i a b e t e s on the h e p a t i c high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n and the p r o s t a t i c androgen r e c e p t o r Tesone et a l , 1980, found a decreased number of 3 a v a i l a b l e (measured by i n c u b a t i o n of c y t o s o l w i t h [ ]R1881 under c o n d i t i o n s of n e g l i g i b l e exchange) and t o t a l (measured 3 by i n c u b a t i o n of c y t o s o l with [ H]R1881 under exchange c o n d i t i o n s ) AR in the p r o s t a t e of 14 day STZ d i a b e t i c W i s t a r r a t s ( c o n t r o l 94.2 + 1 1 . 3 , STZ d i a b e t i c 12.6 + 3.0 fmol/mg p r o t e i n ) , which cou ld be p a r t i a l l y r e s t o r e d by t reatment with i n s u l i n (PZI , 2 I U / r a t , 18.3 + 2.4 fmol/mg p r o t e i n ) or t reatment with t e s t o s t e r o n e (500 (ig/rat s . c . d a i l y , 41.1 + 3.1 fmol/mg p r o t e i n ) . There was no change in a f f i n i t y cons tant ( c o n t r o l 0.13 + 0.01 X 10 9 M, d i a b e t i c s 0.17 + 0.02 X 10 9 M, d i a b e t i c s t r e a t e d with PZI 0.10 + 0.02 X 10 9 M, or d i a b e t i c s t r e a t e d with t e s t o s t e r o n e 0.12 + 0.01 X 1 0 9 M). We found a decrease in b i n d i n g c a p a c i t y of h e p a t i c c y t o s o l i c HALC ABP i n 4 and 10 day STZ d i a b e t i c r a t s ( c o n t r o l 22.51 + 2.48, 4 day d i a b e t i c 9.71 + 1.22, 10 9 1 day d i a b e t i c 8.31 + 1.21 fmol/mg p r o t e i n ) , and no change i n Kd ( c o n t r o l 0.33 + 0 .04 , 4 day d i a b e t i c 0.31 + 0 . 0 5 , 10 day d i a b e t i c 0.36 +_ 0.04 nM). The decreased b i n d i n g c a p a c i t y was not due to decreased temperature s t a b i l i t y of the HALC ABP in STZ d i a b e t i c r a t s ( t a b l e I X ) . No changes i n s p e c i f i c i t y of the HALC ABP were apparent (Tab le X ) . 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 in 4 day STZ d i a b e t i c r a t s was not r e v e r s i b l e by t reatment with PZI (10 U/kg d a i l y , 11.56 + 3.72 fmol/mg p r o t e i n ) ; Toronto i n s u l i n (15 U/kg twice a day, 6.66 +_ 1.30 fmol/mg p r o t e i n ) ; t e s t o s t e r o n e enanthate (1 mg/kg d a i l y , 10.05 + 1.79 fmol/mg p r o t e i n ) ; t r i i o d o t h y r o n i n e (30 ug/kg d a i l y , 9.49 +_ 1.94 fmol/mg p r o t e i n ) ; or oGH a d m i n i s t e r e d by minipump (0.02 U/hour f o r 4 d a y s , 7.16 +_ 1.92 fmol/mg p r o t e i n ) , by m u l t i p l e s . c . doses (30 |ig/dose, 7 doses d a i l y , 7.28 + 2.88 fmol/mg p r o t e i n ) , by m u l t i p l e i n t r a v e n o u s doses by vena cava c a t h e t e r (30 Kg/dose, 7 doses d a i l y , 6.71 + 1.16 fmol/mg p r o t e i n ) , or by t a i l ve in i n j e c t i o n (30 ug/dose, 4 doses d a i l y , 5.41 + 1.93 fmol/mg p r o t e i n ) . A d m i n i s t r a t i o n of PZI (10 U/kg d a i l y ) w i th t e s t o s t e r o n e enanthate (1 mg/kg s . c . d a i l y ) r e s u l t e d i n a p a r t i a l r e s t o r a t i o n in the b i n d i n g c a p a c i t y of the h e p a t i c HALC ABP to 15.7 + 2.91 fmol/mg p r o t e i n . I t shou ld be noted tha t r e s u l t s from the i n i t i a l PZI with t e s t o s t e r o n e enanthate exper iment produced r e s u l t s not d i f f e r e n t from c o n t r o l ; however when r e p e a t e d , r e s u l t s were not d i f f e r e n t from 4 day 9 2 STZ d i a b e t i c v a l u e s . Treatment with PZI (10 U/kg d a i l y ) and t e s t o s t e r o n e enanthate (1 mg/kg s . c d a i l y ) , and with oGH (30 ug/dose , 4 doses d a i l y by t a i l ve in) d i d not r e v e r s e the decrease seen with 4 day STZ d i a b e t i c r a t s (10.12 + 1.02 fmol/mg p r o t e i n ) . T h e r e f o r e , we conc lude tha t r e s t o r a t i o n to c o n t r o l l e v e l s d id not occur with PZI and t e s t o s t e r o n e enanthate a l o n e . B i o B r e e d i n g s t r a i n d i a b e t i c and n o n - d i a b e t i c male r a t s had HALC ABP b i n d i n g c a p a c i t y s i g n i f i c a n t l y l e s s than c o n t r o l W i s t a r males ( W i s t a r c o n t r o l 22.51 + 2.48, B i o B r e e d i n g s t r a i n n o n - d i a b e t i c 11.57 + 2 .23 , B i o B r e e d i n g s t r a i n d i a b e t i c 4.58 _+ 0.73 fmol/mg p r o t e i n ) . The b i n d i n g c a p a c i t y of the B i o B r e e d i n g s t r a i n d i a b e t i c was not s t a t i s t i c a l l y s i g n i f i c a n t l y decreased from the B i o B r e e d i n g s t r a i n n o n - d i a b e t i c (ANOVA and Newman-Keul 1 s range t e s t ) . An i n c r e a s e in number of animals s t u d i e d would be r e q u i r e d to p r o p e r l y t e s t the s i g n i f i c a n c e of the apparent decrease in d i a b e t i c s . There was no change in the Kd va lue of the HALC ABP between W i s t a r c o n t r o l , B i o B r e e d i n g s t r a i n n on-d i a b e t i c, and B i o B r e e d i n g d i a b e t i c r a t s ( 0 . 3 3 +_ 0 .04 , 0.36 + 0 .04 , and 0.49 + 0.32 fmol/mg p r o t e i n , r e s p e c t i v e l y ) . V I I I . N o n - s p e c i f i c e f f e c t s of s t r e p t o z o t o c i n Because of the i n a b i l i t y to r e s t o r e HALC ABP b i n d i n g c a p a c i t y l e v e l s wi th a dose of i n s u l i n which e l i m i n a t e s g l u c o s u r i a in d i a b e t i c male W i s t a r s , the s u g g e s t i o n t h a t the decrease in b i n d i n g c a p a c i t y of the HALC ABP was a d i r e c t t o x i c e f f e c t of STZ was made. A l though d i r e c t t o x i c i t y of STZ i n c l u d i n g d e g r a n u l a t i o n of rough endoplasmic r e t i c u l u m and s w e l l i n g of the m i t o c h o n d r i a has been s t u d i e d i n mice (Laguens e t a l , 1980), ev idence f o r d i r e c t h e p a t i c damage i n STZ d i a b e t i c Wis ta r r a t s i s l i m i t e d to " f o c a l n e c r o s i s " (Junod et al , 1967) . H i s t o l o g i c a l l y , " e s s e n t i a l l y normal" l i v e r s were seen by Warren et a l , 1983. S y n t h e s i s of h e p a t i c and s e c r e t e d p r o t e i n in the l i v e r of STZ d i a b e t i c r a t s i s decreased (McNurlan and G a r l i c k , 1979) . I t i s p o s s i b l e the decrease in the HALC ABP r e s u l t s from t h i s general r e d u c t i o n in p r o t e i n s y n t h e s i s . Breakdown of p r o t e i n i s a l s o i n c r e a s e d 1 day a f t e r i n s u l i n t reatment i s s t o p p e d , and breakdown slows to r a t e s below c o n t r o l r a t e s 4 days a f t e r i n s u l i n i s stopped ( A l b e r t s e et a l , 1980) . I nsul i n - t r e a t e d d i a b e t i c r a t s do not show the decreased ra te of p r o t e i n s y n t h e s i s , nor the i n c r e a s e d p r o t e i n breakdown ( A l b e r t s e et al , 1980). T h i s i s in c o n t r a s t to what i s seen wi th the h e p a t i c HALC ABP s i n c e i t s decrease i s not prevented by i n s u l i n t reatment ( t a b l e X I ) , These f a c t s decrease the p o s s i b i l i t y tha t the decrease seen i n the b i n d i n g c a p a c i t y of the HALC ABP i s a n o n - s p e c i f i c e f f e c t of STZ . A l s o , the h e p a t i c e s t r o g e n r e c e p t o r of the male W i s t a r r a t i s not reduced i n b i n d i n g c a p a c i t y by STZ a d m i n i s t r a t i o n (W.A. Haniuk, 1985) which one would expect i f the decrease in b i n d i n g c a p a c i t y of the HALC ABP was a n o n - s p e c i f i c e f f e c t of STZ. IX. Use of ov ine growth hormone to r e s t o r e h e p a t i c h igh a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n b i n d i n g capac i ty Throughout t h i s study GH replacment was undertaken us ing ov ine r a t h e r than r a t GH, due to l i m i t e d a v a i l a b i l i t y of r a t GH. There i s ev idence in the l i t e r a t u r e f o r a high degree of s i m i l a r i t y between ovine and r a t GH. Sequence s t u d i e s show a high degree of s i m i l a r i t y ( P a l a d i n i et a l , 1 9 8 3 ) . C r o s s r e a c t i v i t y of monkey a n t i - s e r u m to r a t GH wi th oGH in double ant ibody radioimmuno assay occurs (Hayash ida , 1 9 6 9 ) . Ovine GH w i l l no t , however, mimic r a t GH in i n d u c t i o n of GH or p r o l a c t i n r e c e p t o r s in female W i s t a r - F u r t h r a t s , nor i n c r e a s e the serum somatomedin C l e v e l s i n hypophysectomized female r a t s (Baxter et a l , 1984). X. E f f e c t of s t r e s s on the h e p a t i c high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n D u r i n g the course of t h i s s t u d y , i t has become apparent tha t s t r e s s , i n p a r t i c u l a r s u r g i c a l s t r e s s (see e s p e c i a l l y t a b l e X V I I ) , has a g rea t e f f e c t on the measurable amounts of 9 5 h e p a t i c HALC ABP. One p o s s i b l e s o l u t i o n to t h i s problem i s to a l low much longer recovery p e r i o d s a f t e r s u r g e r y . X I . S u b c e l l u l a r l o c a t i o n of the high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n R e c e n t l y the c l a s s i c a l theory of sex s t e r o i d r e c e p t o r f u n c t i o n has been q u e s t i o n e d . Ev idence from a u t o r a d i o g r a p h y , s u b c e l l u l a r f r a c t i o n a t i o n , immunochemistry , and c e l l u l a r e n u c l e a t i o n (King and Greene, 1984, Welshons et a l , 1984, Sher idan e t a l , 1979, S h e r i d a n , 1975) suppor t s the occurence of unoccupied r e c e p t o r s e i t h e r e x c l u s i v e l y in the nuc leus or in e q u i l i b r i u m between the c y t o s o l and the n u c l e u s . Data are not y e t c o n c l u s i v e . A recent d i s c u s s i o n of t h i s s u b j e c t by Wal ters (1985) p o i n t s out tha t p o s s i b i l i t i e s f o r e r r o r e x i s t i n each techn ique used , however s e v e r a l t e c h n i q u e s suppor t the h y p o t h e s i s of n u c l e a r unbound s t e r o i d r e c e p t o r s s t r e n g t h e n i n g the argument. Most s t u d i e s on unbound s t e r o i d r e c e p t o r l o c a t i o n have been done with es t rogen and p r o g e s t i n r e c e p t o r s . Ev idence f o r the e x i s t e n c e of a n u c l e a r unbound AR c o n s i s t s of s t u d i e s where AR were found in the nuc leus but not the cy top lasm in au torad iography of the ra t b r a i n ( S h e r i d a n , 1983). More study i s r e q u i r e d to c o n c l u s i v e l y answer t h i s q u e s t i o n , however we must c o n s i d e r i t s r e l e v a n c e to the i n t r e p r e t a t i o n of our d a t a . The r e c e p t o r l eached from the nuc leus may be l eached randomly or r e s u l t from a decreased a f f i n i t y f o r n u c l e a r b i n d i n g s i t e s . I f the nuc lea r unbound r e c e p t o r l eaches because of decreased a f f i n i t y f o r n u c l e a r b i n d i n g s i t e s , we may be s t u d y i n g a s u b - p o p u l a t i o n of ABP. If we are s t u d y i n g a subpopul a t i o n the o r g i n a l q u e s t i o n of a change in b i n d i n g c h a r a c t e r i s t i c s and s t a b i l i t y or a rea l decrease in numbers of HALC ABP in d i a b e t e s w i l l have to be i n v e s t i g a t e d in d i f f e r e n t ways, and the q u e s t i o n of p h y s i o l o g i c a l r e l e v e n c e of the s u b p o p u l a t i o n as wel l as the general p o p u l a t i o n w i l l have to be answered. A l though much has been s a i d about the n u c l e a r versus c y t o s o l i c r e c e p t o r l o c a t i o n s of an h e p a t i c AR; a s p e c i f i c androgen membrane t r a n s p o r t p r o t e i n on the plasmalemma of can ine and human p r o s t a t e c e l l s ( G i o r g i , 1976), an androgen r e c e p t o r on the nuc lea r envelope of r a t h e p a t i c and p r o s t a t i c c e l l s (Le febvre and Morante, 1982, and L e f e b v r e , 1985) , and androgen b i n d i n g s i t e s in microsomes have been demonstrated and may prove to be very important in the r e g u l a t i o n of androgen e f f e c t s on the l i v e r . I t i s p o s s i b l e t h a t r e g u l a t i o n of androgen e f f e c t s on the l i v e r occurs as a r e s u l t of i n t e r a c t i o n with a l l , one, or none of these h e p a t i c androgen " r e c e p t o r s " . 9 7 X I I . P o s s i b l e f u n c t i o n s of the h e p a t i c h igh a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n The f u n c t i o n of the h e p a t i c HALC ABP i s not immediate ly o b v i o u s . Androgen a d m i n i s t r a t i o n changes the l e v e l of some plasma p r o t e i n s , probably by the m o d i f i c a t i o n of t h e i r s y n t h e s i s in the l i v e r (Anderson and Kappus, 1982) . Androgen a d m i n i s t r a t i o n a l s o i n c r e a s e s the a c t i v i t y of some drug m e t a b o l i z i n g enzymes i n the r a t l i v e r (Kato et al , 1974). S t e r o i d s are b e l i e v e d to take e f f e c t by i n t e r a c t i n g with c e l l u l a r r e c e p t o r s . In examining the aspec t of androgen e f f e c t s on drug m e t a b o l i z i n g enzymes more c l o s e l y we see tha t acute STZ and g e n e t i c ( B i o B r e e d i n g s t r a i n ) d i a b e t e s in r a t s causes a decrease in the sex d i f f e r e n c e s in drug metabol ism in r a t s w i t h i n 4 days (Warren et a l , 1983). These changes are r e v e r s e d i f d i a b e t i c s are t r e a t e d with i n s u l i n . We have i n v e s t i g a t e d the HALC ABP i n STZ d i a b e t i c male Wis ta r r a t s . A l though we found a decrease in h e p a t i c HALC ABP c o r r e s p o n d i n g to f e m i n i z a t i o n of enzyme metabol ism seen in male r a t s 4 days a f t e r i n d u c t i o n of d i a b e t e s , we were unable to prevent the decrease by m a i n t a i n i n g the d i a b e t i c an imals on i n s u l i n a f t e r STZ i n j e c t i o n . T h e r e f o r e i t seems u n l i k e l y tha t the h e p a t i c HALC ABP i s a r e g u l a t o r of sex d i f f e r e n c e s in enzyme metabol ism seen in r a t s . 9 8 FUTURE EXPERIMENTS I . Suc rose d e n s i t y a n a l y s i s of whole and p a r t i a l l y p u r i f i e d c y t o s o l Sucrose d e n s i t y g r a d i e n t s t u d i e s in whole and 50 % ammonium s u l f a t e f r a c t i o n a t e d c y t o s o l have shown r e p e a t e d l y tha t the 8S form of the ABP i s the AR. It would be i n f o r m a t i v e to ana lyze the h e p a t i c HALC ABP tha t r e s u l t s from our 0-50 % f r a c t i o n a t i o n us ing t h i s t e c h n i q u e . I I . E f f e c t s of p r o l o n g e d f r o z e n s t o r a g e of the h e p a t i c HALC androgen b i n d i n g p r o t e i n S t u d i e s on the p r o s t a t i c AR have shown t h a t a f t e r 1 month of f r o z e n s t o r a g e , changes in the i n i t i a l v e l o c i t y of i n t e r a c t i o n with 5a-DHT occur ( F e i t and Muldoon, 1983). A l though no changes were apparent from S c a t c h a r d a n a l y s i s of the h e p a t i c HALC ABP a f t e r up to 60 days of -80°C s t o r a g e , a more r i g o r o u s study i s warranted to determine p o s s i b l e p r o g r e s s i v e degrada t ion of the p r o t e i n with d u r a t i o n of s t o r a g e . I I I . The h e p a t i c high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n of mature female Wis ta r r a t s F u r t h e r i n v e s t i g a t i o n of the i d e n t i t y and c h a r a c t e r i s t i c s of the h e p a t i c c y t o s o l i c R1881 b i n d i n g p r o t e i n in the a d u l t female Wis ta r should be done. In p a r t i c u l a r the presence of a p r o g e s t i n r e c e p t o r in h e p a t i c c y t o s o l should be i n v e s t i ga ted . IV. V e r i f i c a t i o n of the s p e c i f i c i t y of d i a b e t e s c a u s i n g a decrease in number of h e p a t i c high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g s i t e s in male r a t s It would be i n t e r e s t i n g to f u r t h e r c h a r a c t e r i z e 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 seen i n the ABP of d i a b e t i c s . In o rder to v e r i f y the s p e c i f i c i t y of the r e d u c t i o n in the number of HALC ABP b i n d i n g s i t e s in STZ d i a b e t e s i t may be d e s i r a b l e to use 3 - 0 - m e t h y l - D - g l u c o s e . 3 - 0 - m e t h y l - D - g l u c o s e prevents the i n d u c t i o n of STZ d i a b e t e s presumably by p r e v e n t i n g STZ uptake i n t o B p a n c r e a t i c c e l l s . Th i s has been suggested to a l low c l a s s i f i c a t i o n of changes seen in STZ d i a b e t i c animals as " n o n - s p e c i f i c e f f e c t s of STZ" and e f f e c t s of d i a b e t e s . Th i s method depends on the s p e c i f i c i t y of 3 - 0 - m e t h y l - D - g l u c o s e f o r STZ uptake i n t o the pancreas (Ganda et a l , 1976). If 3 - 0 - m e t h y l - D - g l u c o s e i s not s p e c i f i c f o r p a n c r e a t i c c e l l s , but b locks ent ry of STZ i n t o 100 a l l c e l l s of the body, no d i r e c t t o x i c i t y of STZ w i l l be seen e i t h e r . The occurance of decreased b i n d i n g c a p a c i t y of the h e p a t i c HALC ABP in d i a b e t i c ani mal s. whi ch are d i a b e t i c , but not STZ d i a b e t i c , such as B i o B r e e d i n g s t r a i n d i a b e t i c s , would support d i a b e t e s r a t h e r than STZ as the cause of e f f e c t s on the HALC ABP. P r e l i m i n a r y s t u d i e s us ing B i o B r e e d i n g s t r a i n d i a b e t i c and n o n - d i a b e t i c r a t s suggest t h i s i s the c a s e . V e r i f i c a t i o n of these r e s u l t s by us ing a l a r g e r number of an imals would be w o r t h w h i l e . V. R e g u l a t i o n of the h e p a t i c high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n by ov ine growth hormone R e s u l t s from exper iments with oGH a d m i n i s t r a t i o n show tha t the absence of GH or absence of peaks of GH i s not the determinant in the r e d u c t i o n seen i n the HALC ABP. Because of the r e s u l t s of Baxter et a l , 1984, v e r i f i c a t i o n of our r e s u l t s i n which oGH was adminstered us ing r a t GH would be w o r t h w h i l e . A n t i b o d i e s to GH may be e f f e c t i v e in r e s t o r i n g normal trough b lood l e v e l s of GH which may be c r i t i c a l i n m a i n t a i n i n g normal ABP c a p a c i t i e s , depending upon the h a l f l i f e of the a n t i b o d i e s used . If s o m a t o s t a t i n i s r e s p o n s i b l e f o r reduced pu l ses of GH (Tannenbaum, 1981) then s o m a t o s t a t i n a n t i b o d i e s cou ld be used to r e s t o r e peaks w i thout a l t e r i n g trough l e v e l s of GH. 101 V I . De te rmina t ion of the e f f e c t of s t r e s s on the h e p a t i c high a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n Exper iments i n v o l v i n g the d i r e c t a d m i n i s t r a t i o n of C o r t i s o l may p r o v i d e some i n f o r m a t i o n on the e f f e c t s of s t r e s s on the HALC ABP b i n d i n g c a p a c i t y . Exper iments in which gent l ed animals are used may reduce some of the d i f f i c u l t i e s e n c o u n t e r e d . A l so i n c r e a s i n g the l e n g t h of time a f t e r surgery be fore the use of animals in exper iments may be worthwhi1e. V I I . P u r i f i c a t i o n of the h e p a t i c h igh a f f i n i t y - l o w c a p a c i t y androgen b i n d i n g p r o t e i n It would be p r e f e r a b l e to work with a purer system to avo id problems a s s o c i a t e d with small amounts of HALC ABP and c o m p e t i t i o n of endogenous with exogenous l i g a n d s . P u r i f i c a t i o n of an androgen r e c e p t o r from the seminal v e s i c l e of the s t e e r has been done (Chang et a l , 1982). A l s o , the development of a c y t o s o l i c or n u c l e a r exchange assay so tha t a l l androgen r e c e p t o r s i t e s i n c l u d i n g those occup ied by endogenous androgen cou ld be measured, would be d e s i r a b l e . 102 V I I I . I d e n t i f i c a t i o n and l o c a t i o n of the p h y s i o l o g i c a l l y r e l e v a n t androgen r e c e p t o r The f u n c t i o n of the h e p a c t i c c y t o s o l i c AR i s p r e s e n t l y unknown. It i s p o s s i b l e tha t the e f f e c t s of androgen on h e p a t i c p r o t e i n s y n t h e s i s are r e g u l a t e d by another ( m i t o c h o n d r i a l , p lasmalema, or n u c l e a r enve lope) ABP. Th i s s h o u l d be i n v e s t i g a t e d . The l o c a t i o n of the unbound h e p a t i c AR whether n u c l e a r , c y t o p l a s m i c or both w i l l probably be c r i t i c a l i n unders tand ing the mechanism of androgen a c t i o n . As we have seen d i f f e r e n t i a l c e n t r i f u g a t i o n , e n u c l e a t i o n , and a n t i b o d i e s have been used i n an attempt to answer t h i s q u e s t i o n . We have no novel approach to sugges t , but the i n f o r m a t i o n i s impor tant to de te rmine . 103 SUMMARY 1. P a r t i a l p u r i f i c a t i o n of r a t h e p a t i c c y t o s o l u s i n g 50 % ammonium s u l f a t e was found to be b e n e f i c i a l because i t r e s u l t e d in a 2 to 3 f o l d p u r i f i c a t i o n of the HALC ABP ( t a b l e I , whole c y t o s o l 5.86 _+ 0.67 , 50 % ammonium s u l f a t e f r a c t i o n a t e d c y t o s o l 10.37 + 2.34; t a b l e I I I , 50 % ammonium s u l f a t e f r a c t i o n a t e d c y t o s o l 20 .76 +_ 1.49 fmol/mg p r o t e i n ) and no s i g n i f i c a n t (ANOVA and Newman-Keul 1s range t e s t ) change in the Kd (whole c y t o s o l 0.32 + 0 .04 , 50 % ammonium s u l f a t e f r a c t i o n a t e d c y t o s o l 0.30 + 0.02 nM, t a b l e I) o c c u r e d . Ammonium s u l f a t e f r a c t i o n a t i o n a l s o reduced the androgen m e t a b o l i z i n g enzymes, which a l lowed i n c r e a s e d accuracy of d e t e r m i n a t i o n of l i g a n d c o n c e n t r a t i o n , e s p e c i a l l y important i n compet i to r s t u d i e s . 2. The 50 % ammonium s u l f a t e p e l l e t may be f r o z e n and s t o r e d at -80°C, p r e s e r v i n g the HALC ABP wi thout any apparent changes in the HALC ABP assay f o r at l e a s t 60 days ( f i g u r e 2 ) . 3. The HALC ABP was found to be p resent in the immature ( t a b l e V, Kd 0 .40 , 0.07 nM; b i n d i n g c a p a c i t y 9 .20 , 9.57 fmol/mg) and mature female (Kd 0.15 j+ 0.03 nM, b i n d i n g c a p a c i t y 7.04 + 0.94 fmol/mg) and immature male (Kd 0 .10 , 0.15 nM, b i n d i n g c a p a c i t y 9 . 4 5 , 5.40 fmol/mg). Amounts 104 were l e s s than those seen i n the mature male (Kd 0.30 +_ 0 . 0 2 , b i n d i n g c a p a c i t y 20.76 + 1.49 fmol/mg). 4. The d i a b e t i c s t a t e induced by STZ caused a r e d u c t i o n i n the b i n d i n g c a p a c i t y ( t a b l e V I I I , c o n t r o l 22.51 + 2 .48, 4 day STZ d i a b e t i c 9.71 + 1.22 fmol/mg), but no change i n the Kd ( c o n t r o l 0.33 + 0 .04 , 4 day STZ d i a b e t i c 0.31 + 0.05 nM) of the h e p a t i c c y t o s o l i c HALC ABP. 5. The d i a b e t i c s t a t e induced by STZ d id not change the temperature s e n s i t i v i t y and d id not appear to change the s p e c i f i c i t y of b i n d i n g of the h e p a t i c HALC ABP ( t a b l e s IX, and X ) . 6. The lowered b i n d i n g c a p a c i t y of the STZ d i a b e t i c r a t l i v e r HALC ABP cou ld not be r e v e r s e d wi th i n s u l i n ( t a b l e X I , PZI 10 U/kg s . c , d a i l y ; or Toronto i n s u l i n 15 U/kg s . c , twice d a i l y ) , t e s t o s t e r o n e enanthate ( t a b l e X I I , 1 mg/kg s . c , d a i l y ) , t r i i o d o t h y r o n i n e ( t a b l e X I I I , 30 ug/kg s . c , d a i l y ) , or oGH (0.02 U/hour f o r f o u r days by mini pump; or 30 rig/dose s . c , 7 doses d a i l y ; or 30 (ig/dose by vena cava c a t h e t e r , 7 doses d a i l y ; or 30 ug/dose by t a i l ve in i n j e c t i o n , 4 doses d a i l y , t a b l e s XIV, XV, XVI, and X V I I I ) . 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