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

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c. I THE  EFFECT  OF  DIABETES HEPATIC  STREPTOZOTOCIN-INDUCED  ON THE  MALE WISTAR  HIGH C A P A C I T Y ,  ESTROGEN B I N D I N G  LOW  RAT  AFFINITY  PROTEIN  by D'Arcy B.Sc, A THESIS THE  Randall  University  SUBMITTED  IN  REQUIREMENTS MASTER  of  Smith Alberta,  1982  PARTIAL  FULFILMENT  FOR  DEGREE  OF  THE  OF  SCIENCE  i n THE  FACULTY  FACULTY  OF  OF GRADUATE  PHARMACEUTICAL  UNIVERSITY We a c c e p t to  THE  ©  OF  this the  BRITISH  thesis  required  UNIVERSITY  D'Arcy  STUDIES  OF  as  SCIENCES  COLUMBIA conforming  standard  BRITISH  February,  1986  Randall  Smith,  COLUMBIA  1986  OF  In  presenting  degree freely  at  this  the  available  copying  of  department publication  of  in  partial  fulfilment  of  the  University  of  British  Columbia,  I  agree  for  this or  thesis  reference  thesis by  this  for  his thesis  and  scholarly  or for  her  Department  DE-6(3/81)  Columbia  I further  purposes  gain  shall  that  agree  may  representatives.  financial  permission.  T h e U n i v e r s i t y o f British 1956 M a i n M a l l Vancouver, Canada V6T 1Y3  study.  requirements  It not  be  that  the  Library  permission  granted  is  by  understood be  for  allowed  an  advanced  shall for  the that  without  make  it  extensive  head  of  my  copying  or  my  written  1 1  ABSTRACT  Several  parallels  have  been  hydrocarbon  hydroxylase  (AHH)  and s t e r o i d  metabolism)  and t h e  (HCLA)  noted  activity high  estrogen  binding  protein.  age d e p e n d e n c y ,  as w e l l  as p a r a l l e l  HCLA  due  to  gonadectomy  and  hypophysectomy).  evidence  estrogen  that  binding  there  (STZ)  alterations  AHH a c t i v i t y  this  condition  sought  the  binding  with  on t h e  hormonal  protein  At  both  STZ,  binding  four  to  which  treatment reduced  shown  to  in  the  the  are  with  is  the  of  the  HCLA  cause  effects  protein,  the  from  Since  known t o  for  (e.g.  suggested  action  binding  HCLA  post-induction  of  and  estrogen  binding  Hormonal  the  physiological  triiodothyronine,  carried  in  the  out  able  and  to  of  the  HCLA b i n d i n g  testosterone  and  insulin  AHH a c t i v i t y  in  the  replacement  diabetic.  were  in  protein,  Kd v a l u e . normal  diabetes  of  a 50% d e c r e a s e  HCLA e s t r o g e n  capacity  restore  and  AHH and  AHH a c t i v i t y .  mechanism  days  depressed  regimens  a sex the  been  we i n v e s t i g a t e d  approximately  insulin,  binding  Treatment  of  affinity  state.  and t e n  restore  testosterone, of  control  has  diabetes  HCLA e s t r o g e n  this  was  capacity  no a l t e r a t i o n s  all  in  there  undertaken  induced  in  drug  manipulations  a regulatory  on h e p a t i c  streptozotocin in  changes  of  low  include  physiological  is  protein  capacity  It  aryl  (a measure  These  levels  this  various  between  had  was  levels growth  None  of hormone; the  of  restore  with  the  protein. previously  gonadectomized  and  been  diabetic  i  rats,  respectively.  binding rat, of  protein  HCLA  presence  of  unable  species may be  that  binding  Several  were  levels  we c o n c l u d e  the  HCLA  Gail  Bellward,  any  may  What be  Ph.D.  unable  these is  to  were  no d i r e c t  also  estrogen such  binding  remains  HCLA  the  diabetic action  the  rat.  for  the  protein.  in  HCLA  physiological  playing  in  examined  the  in  regulatory  component  that  restore  treatments  on AHH a c t i v i t y  indicating  specific.  protein  Supervi sor  species  detect  binding  D.  with there  a hepatic to  we w e r e  protein  other  examined, rat  Since  any  of  binding  role  the  unclear  i i  at  We the  other  protein HCLA this  time.  i v TABLE  OF  CONTENTS  PAGE ABSTRACT  ii  LIST  OF TABLES  vii  LIST  OF FIGURES  LIST  OF ABBREVIATIONS  viii ix  ACKNOWLEDGEMENT  xi  INTRODUCTION 1.  Diabetes  2.  Hepatic  3.  1 mellitus estrogen  a.  Estrogen  b.  HCLA b i n d i n g  1 binding  receptor protein  Hypothesis  MATERIALS  Chemicals  2.  Animals  3.  Animal  6 6 8 13  AND METHODS  1.  proteins  and  reagents  15 15 15  treatments  16  V  4.  High  capacity,  protein  low  affinity  binding  assay  5.  Determination  6.  Statistical  18 of  protein  content  20  analysis  21  RESULTS  22  1.  Effect  of  possible  2.  Effect  of  streptozotocin-induced  diabetes  3.  Effect  of  insulin  the  diabetic  blood  replacement  in  animal  22 22  29  4.  Effect  of  subcutaneous  5.  Effect  of  hormone  6.  contamination  injections  32  replacement  32  a.  Testosterone  enanthate  32  b.  3,3,5-triiodothyronine  34  c.  Growth  34  hormone  E f f e c t of i n t r a v e n o u s growth administration a. Vena cava c a n n u l a t i o n  horomone  b.  in  Tail  vein  conscious 7.  Other  8.  Stability  administration animal  species studies  40 40  the 42 46 46  v i DISCUSSION  50  1.  Binding  2.  Effect  3.  Hormone  4.  kinetics of  STZ-induced  50 diabetes  replacement  50 53  a.  Insulin  53  b.  Testosterone  53  c.  Triiodothyronine  54  d.  Growth  54  Other  PROPOSED  hormone  species FUTURE  EXPERIMENTS  58 60  SUMMARY  62  BIBLIOGRAPHY  63  APPENDIX  70  1)  I  Saturation  curves  70  v i i LIST  OF TABLES  PAGE Table  I  The e f f e c t of volume on t h e  Table  II  The e f f e c t of S T Z - i n d u c e d d i a b e t e s and i n s u l i n r e p l a c e m e n t on t h e HCLA b i n d i n g p r o t e i n  26  The e f f e c t injections protein  33  Table  Table  Table  Table  Table  Table  III  IV  V  VI  VII  VIII  l i v e r perfusion HCLA b i n d i n g p r o t e i n  ....  of s u b c u t a n e o u s on t h e HCLA b i n d i n g  23  The e f f e c t of hormone r e p l a c e m e n t of t h e HCLA binding protein  35  The e f f e c t of vena c a n n u l a t i o n on the binding protein  41  cava HCLA  The e f f e c t of hormone r e p l a c e m e n t vi a t a i l vein i n j e c t i o n i n the c o n s c i o u s animal on t h e HCLA b i n d i n g  protein  ....  43  O t h e r s p e c i e s examined f o r t h e h e p a t i c HCLA b i n d i n g p r o t e i n  48  S t a b i l i t y study binding protein  49  of  the  HCLA  v i i i LIST 'OF  FIGURES  PAGE Figure  Figure  Figure  Figure  Figure  Figure  1  2  3  4  5  6  Effect volume  of l i v e r p e r f u s i o n on t h e HCLA b i n d i n g  Effect on t h e  of S T Z - i n d u c e d d i a b e t e s HCLA b i n d i n g p r o t e i n  28  E f f e c t of i n s u l i n r e p l a c e m e n t on t h e HCLA b i n d i n g p r o t e i n i n the d i a b e t i c a n i m a l s  31  E f f e c t of t e s t o s t e r o n e and 3,3,5-triiodothyronine replacement in the d i a b e t i c  37  protein  animals  25  E f f e c t of growth hormone r e p l a c e m e n t on t h e HCLA binding protein  39  E f f e c t of t a i l v e i n i n j e c t i o n s i n c o n s c i o u s a n i m a l s on t h e HCLA b i n d i n g p r o t e i n  45  Figure  Al  S a t u r a t i o n c u r v e of c o m p e t i t i v e binding assay, control  Figure  A2  S a t u r a t i o n c u r v e of c o m p e t i t i v e b i n d i n g a s s a y , 4 day S T Z - i n d u c e d diabetic  71  73  1 X  LIST  OF  ABBREVIATIONS  AHH  aryl  BB  bio  Bmax  maximum b i n d i n g  BSA  bovine  serum  cpm  counts  per  DCC  dextran  dpm  disintegrations  DTT  di t h i o t h r e i t o l  EDTA  ethylenediamine  e s t r a d i ol  A  hydrocarbon  hydroxylase  breeding capacity  albumin  minute  coated  l,3,5(10)_  e  s  per  minute  tetraacetic t  r  a  t  r  i  e  n  _  3  F  free  GH  growth  HCLA  high  i .v.  intravenous  Kd  equilibrium  dissociaton  NSB  nonspecific  binding  pmol/mg  picomoles  per  PZI  protamine  zinc  SB  specific  s.c.  subcutaneous  STZ  streptozotoci n  T  3  steroid  charcoal  )  1  7  p_  acid d  1  o  1  concentration  hormone  capacity,  low  affinity  constant  milligram insulin  binding  3 , 3 , 5 - t r i iodothyronine  T.C.D.D.  2,3,7,8-tetracholorodibenzo-p-dioxin  TED  Tris base, ethylenediamine t e t r a a c e t i c a c i d , d i t h i o t h r e i t o l , sodium m o l y b d a t e , g l y c r e r o l  TES  testosterone 4  testosterone To. I  A  -androsten-178-ol-3-one Toronto  regular  insulin  ACKNOWLEDGEMENT  I would Gail  like  Bellward  course parents during  of  this  for  for  to  express  her  guidance  research.  their  my academic  also  attitudes  research  gratitude  and p a t i e n c e  I would  supportive and  my s i n c e r e  like and  training.  to  Dr.  throughout to  th  thank my  understanding  1 INTRODUCTION  1)  Diabetes  mellitus  Diabetes glucose  in  America  has  mellitus  the  been  Complications coronary amongst  artery middle  as  renal  recently  genes  P-cell lack not  of  act  p-cell  such  has  to  in  after  but  genetic  leading  to  cause  and  disease;  blindness;  in  birth  identified  Egypt,  it  realized steps  predispostion  as  factors,  is be  in  the  (probably  with  the  cell  toxins  leading  of  Thus  to  metabolic  or  direct the  diabetes  syndromes.  and e n v i r o n m e n t a l  as  only  to  mechanism and/or  injury.  a group  death  disorders,  was  be s e v e r a l  (such  of  neuropathy;  been  vi a an autoimmune  of  visual  combination  agents  North  vascular  diabetes  a genetic 6),  leading  as e x c e s s i v e  1500 B . C .  amplifying  one d i s e a s e  factors  disease;  appear  in  excess  population.  retinopathy,  as  by  atherosclerosis;  peripheral  regeneration  a variety  amplifying  as  the  peripheral  etiology  diabetes:  prevalence  the  though  environmental  which  actually  reflects  the  There  destruction  of  Even  on chromosome  influence viruses)  of  is  pregnancy,  as e a r l y  that  multifactorial.  two  cataracts,  and t o x e m i a .  development  which  proteinurea;  entity  Its  include:  disease,  during  characterized  2% of  cerebrovascular  complications  very  at  aged d i a b e t i c s ;  disease;  a disease  urine.  diabetes  glaucoma,  weights  and  a disease  estimated  of  hypertension; such  blood  is  It  causes  and t i s s u e  is  and  changes  2 as  chronic  complications  secondary  activity  (Fajans  et  diabetes  and  many m a n i s f e s t a t i o n s  the  the  clinical  concerns  picture  and  of  any  no  one  type  examine  of  These  dog,  the  severity  and  not  of  all  prevalence which  of  the  led  to  insulin  of  it  can  leading  et  C57BL/6J New  obtain  them  create  chemical  take  in  health  in  which  can  others) or  in  the  much  center  methods  animals. on  the  BB  the  The  use  of  to  animal  the  Keeshound  all  vary  they  diabetic.  in  will  done  to  to  create  methods  alloxan.  first It  diabetogenic  was  widely  used  agent as  used  cytotoxic  the  for  research  classical  a  which  chemi c a l s . The  Due  a  trying  been  main  in  develop  maintain  (or  which  of  rats,  to  has  to  diabetes.  become  trying  research  but  genotypic  diabetes  animals  Mordes  used  models  will  in  diabetic  or  develop  These  of  be  a number  and  animal  corresponding  rabbits,  colony  biological  developed  are  white  arise  research  1985)  should  Zucker  ape.  the  Like,  precisely  There  mice,  black  see  1982;  model  different  reviews  pathologic,  pathogenesis  from  state  the  Zealand  animals  al., of  many  (for  spontaneously  spontaneously  been  available  capable  which  and  of  has  disease.  Celebes  colony  diabetic  is  the  difficulties  have  The  one  phenotypic,  hamster,  and  being  human d i a b e t e s  include:  the  to  it  diabetes  model  available  Chinese  make  1981; Grodsky  specific  expressions models  into  diabetes  Rossini,  since  1978).  insufficient  today.  Research models  al . ,  to  was  diabetogen  3 until  streptozotocin  al.,  1960).  Studies  showed t h a t (Hoftiezer  it and  toxicity)  were  of  al. ,  diabetic  states  tissue  metabolic  in  al . ,  the  These  factors as  factors,  alloxan  main  mellitus, the  (Arison  et  importantly,  more  closely  (Mansford  most  weight  al . ,  the  validity  model  for  the  for  Opie,  resemble  to  of  highly  a  series  the  dose in  those  seen  1968)  and  those  seen  the in  1985).  STZ-induced  disease.  Due t o  by STZ as  experimentally  strains  1967 ;  (Kloppel,  replaced  alloxan  changes  resemble  mellitus  been  the  quickly  P-cell  produce  and  clearly  largely  agent in  (i.e.  by v a r y i n g  a relevant  except  lesions  et  general  severity  human d i a b e t e s  diabetogenic  had a l o w e r  graded Most  (Vavra  than  to  by STZ  has  rate  possible  mellitus  give  diabetes  it  1959  diabetes,  mortality  1973),  patterns  of  induce  in  was  1969).  produced  insulitis  It  of  human d i a b e t e s  lesions  STZ t o  reproducible  1969).  et  introduced  characteristic  more  (Junod  was  a lower  Carpenter,  and t h e  et  using  produced  toxicity,  Junod  (STZ)  induced  resistant  to  these the  diabetes  STZ,  such  rabbit. The  diabetogenic  destruction 1981).  of  the  inverse severity  et  induced  al.,  and d e c r e a s e d 1974;  relationship of,  state  the  STZ i s  8 - e e l Is  hypoinsulinemia,  hyperketonemia, (Montoya  of  pancreatic  The d i a b e t i c  hyperglycemia,  action  Chen  between  diabetogenic  to  (Mordes  by STZ  is  its et  selective al. ,  marked  by  hyperlipemia,  growth  et  due  al.,  and body 1982).  sensitivity properties  to, of  weight  There  is  and  the  STZ  an  and t h e  age  as  4 of  the  animal  evidence female  (Masiello  that  in  multiple  the  both dose  Besides  male  single  P-cell  destruction.  reduced to  to  male  rats  rats  show an i n c r e a s e  levels  (Warren et  (Learning  is  mainly  of  of  ketosis.  to  a decrease  releasing  et  in  the  male  those al., in  level  decrease  in  the  of  growth  storage  the  is  STZ  is  in  of  et  al.,  to  of  with  hypothalamic  (Mi t s uma et  of  al.,  1984).  seen due t o et  al. , levels  pituitary,  pointing release  to  of  growth  pattern  of  be  due  fall, and  the  there  is  a  to 1982;  deficient  Jennings, in  the  synthesis  However,  growth  severity  thyroxin,  A decrease  1984).  to  increase  thyroid  increases  al.,  the  male  thyrotropin  and  levels  thyroxin  female  normal  reported  pituitary  to  1981)  testosterone  also  due  diabetic  STZ-treated  correlates  plasma  hormone  insulin  spontaneously  is  and  significantly  (Baxter  This  the  affects  and 3 , 3 - d i i o d o t h y r o n i n e  normal  The  and  causes  (1981) measured  deficiency.  1982).  than  1980)  serum t e s t o s t e r o n e  release  et  (Bluet-Pajot  thus  in  also  1978).  state,  animals seen  conversion  hormone  al . ,  al . ,  3,5-diiodothyronine  Ortiz-Caro  et  a decrease  dysfunction  3 ,3 , 5 - t r i i o d o t h y r o n i n e 1984;  is  origin  As w e l l ,  of  et  is  senstive  1983), while  a1 . ,  3 ,3 , 5 - t r i i o d o t h y r o n i n e , plasma  (MacLeren  There  hormone which  alterations.  more  There  Serum t e s t o s t e r o n e  adrenal Thyroid  is  a diabetic  STZ-treated similar  1979).  (Rossini  secretion.  in  levels  dose  producing  and  al . ,  animal  regimens  supply  et  release and  Tannenbaum  hormone  in  the  hormone  release  as  the  growth  hormone  in  the  5  male  diabetic  trough' level'  release  also  to  circulating seen  levels  of  et  mitochondrial  swelling  of  not  whether  or  the  1980).  Its  include  decreased  acid  also  content,  diabetes been  and  Warren  protein  distribution  et  al.  in  others)  also  et  al. ,  Stohs  1983).  metabolism treatment  been et  (Reinke  1979).  changes  the  STZ-induced  diabetic  authors  was t o  effect  et  drug  between the al. ,  Cook,  the  changes citric  proteins,  and  Herrera, in  1978;  and t h e  These in  (Reinke  et  1980;  Peng  drug  sexes. and  Reinke were  the  region.  make  biochemical  metabolism  animal  al. ,  and d e c r e a s e s  the  seen  in  P-450  produces  et  alterations  and  abnormalities  are  independent  hepatic  content,  cytochrome  reverse  the  and  (Languens  1979 ; Past  to  These  reported  differentiated  It  cristea,  (Montoya  by o t h e r  able  the  and p r o l a c t i n  liver.  of  coA  seen  was  in  in  concentration,  some bands  al . ,  Decreases  other  in  Overall,  less  that  acetyl  the  patterns  reticulum  produced  glycogen  (1983)  (increases  1978;  loss  P concentration  alterations  al.,  is  increased  1974).  somatostatin  on the  the  reported  weight,  1976;  of  hormone  and  'continuous  release  endoplasmic  with  'peak  1982).  effects  rough  and DNA p h o s p h o l i p i d  have  al. ,  direct  of  normal  in  1981).  luitenizing  degranulation  its  female  levels  (Tannenbaum,  (Perez-Diaz has  the  alteration  increased  animal  STZ a l s o  to  from  (Tannenbaum and M a r t i n ,  This  be due t o  changed  release  pattern  1981).  STZ-diabetic  also  is  (ultradiene)  Tannenbaum, seems  rat  et  Insulin metabolic al. ,  similar  spontaneously  in  6 diabetic  animals  2)  Hepatic  a)  Estrogen The  rat  estrogen  to  uterus:  the  non-steroidal  (fmol/mg)  focusing  profile  Eisenfeld  Wrange found  et in  levels  other  1977;  been  such  as  the  human The  levels 1980; in  hepatic  et  the  rabbit,  (Duffy  both  et  Beers  uterus 1982).  liver  estrogen  of  female  et  al. ,  1981)  receptor  both  in  M) and  in  the  low  isoelectric  Viladiu  et  al . ,  has  also  been  at  lower  Rosner,  1977;  but et  estrogen sexes  were  of  (Eisenfeld  al. , receptor  other et  has  species  al . ,  1 9 77 ) ,  1978).  and  estrogen  found  receptor  The  receptor  animal  which  and  1975 ;  heart)  the  sucrose  (Eisenfeld  monkey  male  post-pubertal  on  (10  and  kidney,  and D u f f y ,  showed  a similar  al . ,  (e.g.  green  receptor  receptor  and  estrogen  al.,  This  steroidal  The  or  in  affinity  1976;  liver  in  Thompson  the  al . ,  the  for  high  be found  S region  estrogens;  tissues  hepatic  in  for  1980).  found  8-9  (Chariness  Singletary  also  and  in  the  to  characteristics  estrogen  specificity  et  al . ,  than  in  estrogens;  capacity  1975;  receptor.  'classical'  gradients;  protein  and b i n d i n g  sedimentation  density  1983).  proteins  binding  estrogen  properties  identical  binding  estrogen  was t h e  physical  al . ,  receptor  first  liver  (Warren et  is  rats and  in  similar  (Powel1-Jones reaches  (Rumbaugh both  found  et  sexes  its  al., is  et  al. ,  highest 1983).  under  level The  7 multihormonal the  levels  of  and R o s n e r , al.,  regulation. receptor  1977 ; Eagon  1981), while  receptor  levels  in  1981  The e v i d e n c e  probable 1981  a;  hepatic  complexes estrogenic but  in  retention 1978; The  levels  of  estrogen  al.,  in  renin  levels  1983),  of  and  mRNA s e q u e n c e avian  liver  arisen present  on t h e  very  apo  low  whether  al. , the  cytosol  nuclear  a;  et  al . ,  mechanism of  as  Norstedt  has  et  complexed  a 1. ,  slower et  and E i s e n f e l d , have  been  density  et  levels  density  al. ,  the  is  (i.e.  is  the  al. ,  with  resides  et  specific  1ipoprotein-11  receptor truly  nuclear  (Thompson  estrogen it  and  19 7 6 ) ,  debate  if  females,  correlated  of  an  1980).  lipoproteins  the  been  it  with  and  1976 ; Aten  (Eisenfeld  low  is  has  where  both males  al.,  in  cytosol  Recently  membrane  in  the  1978).  or  the  glucocorticoids  the  process  receptor  very  in  1981  (Norstedt  19 75 ;  in  in  Dickson  an i n c r e a s e  for  the  it  et  substrate  (Snow et  as t o in  1980;  al.,  translocation  occurs  (Eisenfeld  al. ,  et  (Beers et  a decrease  towards  al . ,  translocation  longer  causes  receptor  after  the  rats  in  1983 ) .  chromatin This  increase  Powell-Jones  and p r o l a c t i n  et  nuclear  et  plasma  or  al. ,  Aten  increases  hormone  an  female  regulatory  undergo  ligand.  males  1980;  points  estrogen  to  with  et  and  (Norstedt  the  (Chamness  Gustafsson  demonstrated  for  growth  agents  The  al . ,  causes  adrenalectomy  receptor  with  male  sexes  as does  estrogen  combination  et  both  1982),  hepatic  both  hypophysectomy  Eriksson, a).  in  Gonadectomy  in  has  actually in  accepted  the model  nucleus of  8 receptor-1igand to  the  nucleus  of  experimental  Greene,  1984;  question end  to  result  from t h e Not  all  complexing to  with  Schrader,  answer,  liver  but  the  are mediated  hepatic  responses  by e s t r o g e n  interacting  with  chromatin  with  1983).  receptors  (Pietras  and  and S z e g o , 1971;  that  1974),  to  has  et  these  translocating  on an 1984;  artifact King  and  important not  a series  change  of  by e s t r o g e n  the  responses  receptors.  seen due t o  estrogens  receptor-1igand  complexes  that  associated  with  1980),  and m i c r o s o m a l  al . ,  the  Lax bind  membrane  membranes  and M i y a j i , cause  also  plasma  (Blyth  can  19 82 ;  estrogens  membranes  could  et  lysosomal  1977 ; Yamada  membranes  an  will  found  1979;  al. ,  is  (Tamulevicius  been  are  Szego,  Helton  Binding  It  al•,  it  do e l i c i t  for  al. ,  way  then  based  et  This  either  be a c c o u n t e d  et  cytosol  (Gorski  1984).  estrogens which  the  chromatin  procedure)  that  of  bind  in  et  (Hirsch al. ,  1982).  effects  by  altering  2+ intracellular rearranging  Ca  concentrations  some b i o l o g i c a l l y  important  membrane  or m i c r o s o m a l  system.  b)  capacity,  affinity  High  Besides of  estrogen  referred binding  to  estrogen  binding as t h e  high  binding  receptor  protein  in  components  capacity,  rat  low  of  the  protein  there  male  or  is  a second  liver  affinity  which  class shall  (HCLA)  protein.  The groups  the  low  and d i s t r i b u t i o n ,  to  HCLA b i n d i n g have  binding  protein  has  been  characteristics  reported which  by  show  several lower  be  9 affinity  and h i g h e r  systems.  Values  -8 10" M , al.,  which  binding  1980);  capacity have  been  Kd = 1 0 " - 1 0 " M ,  mol/mg  protein  binding  capacity -8 Kd=10" M,  and  seen w i t h  reported  are:  mol/mg  binding  8  (Rogerson  those  -12 10"  capacity 7  than  Kd=10" 7  protein  capacity  Eagon,  receptor  1984);  10"  (Eagon  et  1 3  Kd=10" M, 7  -15 1978);  (Smirnov  et  10  mol/mg  p r o t e i n ( D i c k s o n et a l . , -1 1 capacity 10" mol/mg p r o t e i n  binding  al. ,  1977);  Kd = 1 0 " M ,  binding  7  capacity  -1 ? 10  mol/mg p r o t e i n  Differences  in  methodologies density  the  values  used  to  gradients,  Scatchard  (Warren,  reported  measure  gel  analysis  of  multi-point  differences  in  cytosol,  and  analysis  the  binding  protein  density  gradients  1980;  estrogen et  al. ,  1980). peak  analysis  of  and f e m a l e 1980 ;  in  affinity  of  being  estradiol,  single  point  analysis,  al.,  from a d u l t  et  1981).  and  for  binding  prepare  obtained. of  compared in  the  protein  to  8-9  The  al. ,  the  S region  also  shows  not  female  or  prepubescent  The  Eagon  HCLA  seen  et  steroidal  approximately  estrogens  in  the male  al . ,  binding  and some a n d r o g e n s  (Eagon  up as  is  1978;  HCLA  sucrose  1977 ; Eagon et  as  the  which  al . ,  estrogens  used to  S region  et  estrone)  sucrose  3-4  1980)  various  (e.g.  the  et  the  protein  data  filtration  highest  to  the  (Dickson  binding  may be due  competitive  sediments  al . ,  1983).  of  HCLA b i n d i n g  gel  cytosol rats  al. ,  which  The  Thompson  capable  et  Finlayson,  buffers  in  (Smirnov  receptor  a unique  the  sediments  Powell-Jones  the  filtration,  curves),  in  1982;  protein  with  the  (estriol,  10-20 t i m e s  less  for  is  1 0 the  androgens  which  bind  (2cc-hydroxytestosterone,  5a-androstane-3a,17p-dio1, (Miroshnechenko The  important  estrogens groups,  of  The the  al.  Dickson  are  of  which  of  the  ( 1978)  Other  HCLA b i n d i n g  protein  1980;  reported rat  and t h e  those  seen  (1980) the  this  in  found  of  normal  but  of  male  protein the  in  during  in  neonatal  levels  the  seen  not  livers  et  while  detect  al. in  were  protein  surge  seen is  the  protein  the  in  et  the  seen  is  allowing reach  in  (days  male  for  has  to  al.  not  development and  et  immature  similar  cue  This  mature m a l e ,  animals  the  puberty  which  7-12.  (Eagon  was  after  the  (1981)  Powell-Jones  evident  when t h e s e  higher  levels,  liver  were  castrates  much  Thompson  to  et  preventing  are  1983).  hundred-fold  Thompson  days  al.,  in  female  surge  both  binding  female.  been u n a b l e  HCLA b i n d i n g became  et  1984).  hydroxy  the  protein  a two  female.  (for  17-P  difference  their  HCLA b i n d i n g  castration  binding  1983).  testosterone  the  and  decreases  mature  testosterone  imprinted  neonatal  have  the  found  the  the  This  neonatally  treatment  in  mature  male,  time.  HCLA  the that  due t o  expression  by  groups  differences  levels  immature  34-42)  sex  3-a  t h e mature  reported  Finlayson,  that  in  Eagon,  binding  HCLA b i n d i n g  a ten-fold  difference.  al.,  the  and  (Miroshnechenko  male t h a n  al .  for  sharply  steroid  reported  et  Rogerson  androgens)  levels  (1981)  1983;  groups  the  mature  al. ,  functional  removal  affinity  in  and  et  5a-dihydrotestosterone)  at  the  been demonstrated of  the  testosterone expression adulthood  of (Sloop  1 1 et  al. ,  1983 ; Thompson  androgens binding adult in  are  the  neccesary  protein  male  levels  resulting  HCLA b i n d i n g  In  addition  rats  resulted  expression protein the  in  of  female)  agent  playing  that  (male  in  1983).  levels  was  ectopic animal  also  ectopic  was  identified  of  1980;  Lucier  (1983) in  the  which  with  certainty  Growth  hormone  et in  The main seemed t o  HCLA b i n d i n g but and  of  the  et  the  binding  be  the  in  the  HCLA  (1981) to  on HCLA  binding  a further  (1983)  when  50-80% animal  protein an  hypophysectomized secreted  responsible levels,  evidence  prolactin  in  al., be  seen  hormone(s)  protein the  in  HCLA b i n d i n g  al.  an  female  hypophysectomized  repression  implanted  and  to  al.  reported  by Rumbaugh  female).  seems  administration  decrease  increased  Lucier  et  the  requires  HCLA  and  differences  had no e f f e c t  capacity  seen  pituitary,  feminization  hormone.  or  While  male  of  expression  male  (i.e.  hormone  sex  for  differences  protein  al.,  hormone  This  sex  HCLA  1983).  also  adult  in the  et  the  Finlayson  pituitary (male  in  animals  and f e m a l e ) .  the  (Finlayson,  of  expressed,  by c a s t r a t i o n  protein  of  Growth  levels.  binding  Once of  dependency  binding  abolition  growth  levels,  decrease  levels  HCLA b i n d i n g  hypophysectomized protein  HCLA  a role  protein  maintanence  androgen  (Powel1-Jones  Finlayson,  reported  the  the  1983 ) .  a testosterone-reversible  were d e c r e a s e d  1981;  binding  the  Hypophysectomy  the  levels  Lucier,  as d e m o n s t r a t e d  in  the  pituitary.  for  protein  to  and m a i n t a n e n c e , intact  and  are  has  points  for not to  secreted  by  the been growth  in  the  an  1 2 largest 1980)  amounts  and  effect  since  et  hormone The  effect Its  level.  receptors al. ,  the  suggested  to  androgens  disposition  the  to  for  (Norstedt  et  of  the  for a  the  feminizing  a novel  male  al.,  the  exogenous  have  of  of  is  strengthens  not  concentrations  1983).  pituitary  can  is  growth  levels  responsible  hormone system  al.,  protein  hypophysectomized  the  between  of  1978;  et sex  HCLA b i n d i n g uptake  concept.  rats  leads  to  prolactin 1981  b;  al.,  mechanism  metabolites)  1982),  steroids  that  the for  protein  Norstedt  et  thereby  and t h e and t h e  and t h e i r al.,  HCLA the  have  and c o n c e n t r a t i o n  receptors  Si n g l e t a r y  possibility  modifying  the  (and t h e i r  (Miroshnechenko  al. ,  binding  liver  be:  distribution  et  growth  as  termed  1984).  Functions  and  That  is  be  which was  Mode et  1 9 8 3 ) , which  metabolism)  must  identified  ectopic  al . ,  steriod  there  administration  hormone  increased)  in  1980;  from the  growth  on a s t e r o i d  later  et  feminizing  released  on HCLA b i n d i n g  (Finlayson,  that  (i.e.  was  upon t h e  administration  female  et  seen  (e.g.  concluded  being  Colby,  secreted  hormone  decreased  was  factor  effect  hormone  probability  parameters it  and  1976 ; G u s t a f s s o n  had no m e a s u r a b l e  Feminotropin  (Rumbaugh  same as t h a t growth  1977)  "feminizing"  feminizing  growth  hepatic  al.,  feminotropin.  al . ,  prolactin  on o t h e r  (Eneroth another  (Lam et  estrogens  regulating of  There protein  translocation  of  the  metabolism  and  metabolites  1983).  binding  of  enzymes binding  been  (Dickson  is  also  may  the  serve  estrogen  as a  1 3 receptor  (Powel1-Jones  show t r a n s l o c a t i o n experimental indicator  use  for  toxicants,  al.,  itself  for  the  et  the  (Dickson  initial  in  adult  although  et  al. ,  HCLA b i n d i n g screening  as d e m o n s t r a t e d  (feminization)  1980)  by the  males  male  mercury,  1 , 2 - d i b r o m o - 3 - c h i o r o p r o p a n e , and al. ,  1984;  work  in  Lui,  An  may be  1985 ;  as  an  levels  (and m a s c u l i n i z a t i o n toxicants  not  reproductive  decreased  exposed  et  to  of  does  1978).  protein  females)  (Lawrence  neonatally  it  such  as  in  adult  methyl  chlordecone  Lawrence-Domey  and  Lui,  1985).  3)  Hypothesis Previous  1983)  has  estrogen  shown a number binding  hydroxylase observed These  sexual  of  the  gonadectomy the  capacity  activity; in  the  in  differences  HCLA b i n d i n g  produces of  the  of  growth  hypophysectomized  binding  activity  in  between  levels  of  male  rat,  the  drug  and  sex  protein  Finlayson,  aryl  steroid  difference  and  HCLA b i n d i n g  AHH  the  abolishes  Since  the  HCLA b i n d i n g  hormone male  protein  or  female  rat  decreases there  is  to  in  and  the  metabolism. in  the  decrease  in  AHH  in  seen  AHH  the  causes  the  hydrocarbon  differences  protein  administration  HCLA  activity;  and  sex  the  reflecting  a testosterone-reversible  and f u r t h e r  protein.  1982;  correlations  and t h e  hypophysectomy  AHH a c t i v i t y  (Warren,  an age dependent  capacity  activity;  lab of  protein  (AHH)  include:  levels  our  a reduction  capacity  a reduction  of  of the  the sex  in HCLA  differences  in  drug  diabetic  animal  mediated  in  part  by t h e  investigated  HCLA  estrogen  also  in  estrogen  in  binding  the  effects protein  protein  investigated  physiological  Testosterone  doses  enanthate,  3 ,3 , 5 - t r i i o d o t h y r o n i n e Toronto  regular  growth  hormone  levels  of  insulin four  the  ultradiene  pattern  the  ultradiene  pattern  and t r o u g h s  of  the  of at  STZ-induced periods  in  pathological  effects the  of  zinc  once  was  administered  twice  need t o need t o  ultradiene  had t o  the  d a y s ) . We the  HCLA  condition  and  replacement  a day. a day.  With  be e x a m i n e d :  mean  be m a i n t a i n e d ,  be p r e s e n t , be p r e s e n t , pattern  the  insulin,  administered  to  where  on  animal.  were  GH need  being  diabetes  hormonal  STZ-diabetic  protamine  of  STZ-  p r o t e i n . We  (4 and 10  mechanism  this  the  may be  control  the to  in  binding  time  possibilities  circulating  this  were e v i d e n t  hormonal  binding  therefore  the  that  HCLA e s t r o g e n  AHH a c t i v i t y  sought  metabolism  we h y p o t h e s i z e d  thus  changes  and s t e r o i d  need  the  the  peaks  troughs  or  both  to  be  the  of  of peaks  maintained.  MATERIALS  1)  Chemicals The  and R e a g e n t s  following  from Sigma C h e m i c a l charcoal, blue  bovine  chemicals Company  acid  and r e a g e n t s  (St. Louis,  serum a l b u m i n  G, D L - d i t h i o t h r e i t o l  tetraacetic  AND METHODS  (BSA),  (DTT),  (EDTA),  (T )  1 7 P - e s t r a d i o l , Trizma® b a s e .  Estradiol, were  obtained  Dextran  Sweden).  Institute  of D i a b e t e s ,  Protamine  zinc  purchased  at  and Company, reagents  2)  were  streptozotocin  [6,7- H(N)]3  scintillation  Nuclear  Corporation  Fine  Digestive,  and T o r o n t o  retail  C h e m i c a l s AB  Toronto,  Ont).  of a n a l y t i c a l  Model 2001  Alta,  of t h e  and K i d n e y  insulin  outlets  (Palo  was a g i f t  (Connaught  other  C a . ) . Ovine  National  Diseases. Labs)  as was t h e Tes-tape® All  cocktail  (Boston, Ma.).  from P h a r m a c i a  ( 1 . 5 I.U./mg)  brilliant  ethylenediamine  Alzet® o s m o t i c - m i n i p u m p s ,  from A l z a  hormone  activated  3 ,3 ' , 5 - 1 r i i o d o - L - t h y r o n i n e  and B i o f l u o r  from New E n g l a n d  obtained  growth  Ci/mmol  T-70 was o b t a i n e d  (Uppsala, were  40-60  enanthate,  obtained  Coomassie  sodium m o l y b d a t e ,  testosterone  ,  Mo.):  disodium  (STZ), 3  were  chemicals  were  (Eli  Lilly  and  quality.  Animals Male W i s t a r  rats  and male  DBA/2 mice  Canadian  Breeding  weighing  325-375  weighing  weighing  Farms  300-350  20-30  (Montreal,  grams were  g r a m s , male  grams were P.Q.).  obtained  from  C57BL/6  obtained  Male local  guinea  from pigs  suppliers  1 6 (U.B.C  Animal  animal  room on Lobund® b e d d i n g  Paxton,  Care).  I I . ) under  and t e m p e r a t u r e food  (Purina  Ltd,  Woodstock,  3)  (22°C).  animal  citrate under  allowed The  buffer.  hormones  (30 fig/kg  Before  insulin  were  i n normal  was a d m i n i s t e r e d hormone  four of  to  Canada  with  saline  s.c.  seven  times  were  after  once  only  shipment.  a day:  testosterone  o i l ) , triiodothyronine at pH 8 . 5 ) .  twice  s.c.  Toronto  a day (15 u n i t s / k g ) .  (GH) was d i s s o l v e d  M-g/injection  rats  prior  in  vehicle  to e q u i l i b r a t e  in corn  s.c.  or t e n days 60 mg/kg  ( P Z I ) (10 u n i t s / k g ) ,  1) a d m i n i s t e r e d  in  normal  i n a dose  o f 30  saline  (pH  p e r day at 6:15 a . m . , 10:15 a . m . ,  p . m . , 6:15 p . m . , 9 : 0 0 p . m . , 1 1 : 4 0 p . m . , and 2:40 a . m . ;  f o r continuous  minipumps ether  into  of  injection,  injected  = 8.5) and:  of  access  ad l i b i t u m .  injected  3-4 days  ( T E S ) (1 mg/kg  2)  free  Purina  injection  were  anaesthetic.  enanthate  2:15  allowed  Ralston  vein  Controls  a minimum of  growth  Processing L t d . ,  was a d m i n i s t e r e d  zinc  Ovine  separate  (6 a . m . o n , 8 p . m . o f f )  O n t . ) and t a p w a t e r  protamine  insulin  in a  Treatments  following  (Tg)  (Paxton light  Chow,  use v i a a t a i l  ether  housed  They were  Laboratory  Animal  were  controlled  Streptozotocin to  Animals  were  implanted  aneasthetized  t h e pumps  filling  infusion  of GH, Alzet® Model  subcutaneously  rats.  in the r o s t r a l  The GH s o l u t i o n  and t h e pumps were weighed  to ensure  complete  filling.  2001  was l o a d e d  before  The pumps  half  and  had a  after life  1 7 expectancy  of  0. 06  uL/hr  (equivalent  1. v .  injection  conscious 5:00  method the  and  a pumping to  four  and 9:00  set  at  a dose  rate of  GH was a d m i n i s t e r e d  animals  p.m.,  related  7 days  of  times  p.m.)  in  experiments  had  TES  and  same i n j e c t i o n  per  a dose  pattern  30  the  For  vein  a.m.,  in  1:00  p.m.,  ng/injection.  same GH i . v .  to  and dose  tail  (9:00 of  uL/hr +_  units/hr).  vi a the  day  added  1.04  0.02  used t h e  PZI  of  injection  treatment  as t h e y  A  regimen  were when  in  given  alone. Rats  were  following  cannulated  standard  anaesthetized made t h r o u g h located where  and  the  the  dorsally  at  skin in  of  the  the  the  inserted  animal  was t o muscle  neck  back.  be p l a c e d .  region  The  to  cannula to  checked.  the The  the  vena  tissue  for  anchoring.  over  the  was  placed  removed in  wound,  sutured  topically  cava  to  the  exit other  area pushed  allowed  to  under  the  flow  the  tied  exit  to  solution  were layers was  surgery.  treatments  recovery  was  nearby  and s k i n  from t h e  through  cannula  antibiotics  muscle  for  and  end of  halothane,  cava was  placed  Lidocaine  cage  was  and  point  Topical  pain  the  a cavity  and l o o s e l y  alleviate  over  spine  incision  vena  A trocar  was t h e n  closed.  an i n d i v i d u a l  were  The  and  create  and the  from t h e  Rats  layers.  underneath  beside  and t a k e n  was  carefully  applied  and  the  passed  anaesthesia  and a midabdominal  and muscle  into  sprinkled were  back  trocar  cannula  muscle  sutures  halothane  procedures.  halothane  skin  cannula  through  surgical  with the  under  and  The  started, use.  1 8 4)  High  capacity,  low  affinity  Rats  and mice  were  stunned  killed  by d e c a p i t a t i o n .  cervical  dislocation  perfused  v i a the  50mM T r i s ,  molybdate,  and  excised  recorded. time  of  1.5mM  into  killing minced  samples  minutes  using  at  (Needham  4°C  Hts.,  resulting  Ma.)  supernatant  minutes  at  4°C  Beckman  L5-50  (to  supernatant protein  Instruments  was  removed  concentration  Bradford  protein  ( 1 : 1 0 w/v)  assay  at  at  with  Palo  mg/ml  method  10  Company  centrifuge. for  via  1976)  a  angle  Ca.).  determined  The to  a  the  and  placed  i ce. The determined binding cytosol,  binding  by S c a t c h a r d  curves. 10  characteristics  The  (1949)  3  estradiol  analysis  incubation  uL [ H ] - e s t r a d i o l  of  mixture  of  were  competitive  contained  (10 Ci/mmol)  10-200  The  30  using  TED b u f f e r  (Bradford,  Livers  for  65 f i x e d  Alto  the  buffer  10,000g  fraction)  with  at  After  100,000g  a type  were  weight  TED  Equipment  cytosolic  Corp.,  1-3  in  refrigerated  and d i l u t e d  of  livers  diabetes.  homogenizer.  B-20  the  The  glycosuria  of  were  20mM sodium  for  presence  and  buffer,  to  test  head  Livers  TED  4°C.  centrifuged  ultracentrifuge  (Beckman  via  and t h e  was c e n t r i f u g e d  obtain  killed  0.5mM DTT,  International  Model  the  dry  tissue  an  to  cold  at  assay  blotted  the  were  were  pH 7.5  and homogenized  a Potter-Elvehjem  a blow  ice  EDTA,  used  confirm  pigs  with  TED b u f f e r ,  to  with  binding  by d e c a p i t a t i o n .  10% g l y c e r o l ,  homogenization  rotor  vein  Tes-tape® was  were t h e n using  followed  portal  containing  then  Guinea  protein  500 nM  ul (in  on  1 9 absolute excess out  ethanol)  of  for  in  unlabelled  90 m i n u t e s  bound  steroid  was  liL of  dextran  T-70  (DCC)  in  at  4°C.  Following  at  4°C  a type  sampled  JS  (0.5  System, II.).  4.2  using  rotor.  The Mark  The  so d a t a  Mark  (Searle  III  was  could  of  J6-B  the  interfaced be  minus  DCC t h e at  counted  recorded  of  500  samples  were  150Og f o r  10  centrifuge were  liquid for  bound  Scintillation  Inc.,  with  and  the  supernatants  Liquid  Analytical  carried  (0.5% w/v)  refrigerated  were  fold  addition  10 mL B i o f l u o r  III  a 100  were  charcoal  resulting  Samples  a Searle  6880  by  previously  addition  a Beckman  of  was t e r m i n a t e d  by c e n t r i f u g a t i o n  cocktail.  Model  computer  coated  mL) and mixed w i t h  scintillation estradiol  using  reaction  prepared  the  absence  from f r e e  (0.05% w/v) as  or  Incubations  The  separated  and DCC s e d i m e n t e d  minutes with  presence  estradiol.  TED b u f f e r ,  molybdate. mixed  the  Des  Plains,  an A p p l e  directly  to  Plus  disk.  II All  p o i n t s were a s s a y e d i n d u p l i c a t e . The t u b e s w h i c h c o n t a i n e d 3 [ H ] - e s t r a d i o l w i t h o u t c o m p e t i t o r measured t o t a l binding 3 (TB),  while  competitor  the  tubes  with  represented  the  both  [ Hj-estradiol  non-specific  and  binding  (NSB)  present. Analysis counts  per  converted  of  minute to  (cpm)  data  efficiency  tritium  was 47%. which  of Thus  proceeded 3  recorded  disintegrations  percent  efficiency,  binding  the  per  for  dpms were  was d e t e r m i n e d  (dpm)  each  on  the  for  on t h e  sample  The were  based  Efficiency  calculated for  follows.  [ H]-estradiol  minute  instrument.  as  basis  from  of  2 0 previously  prepared  converted The  to  protein from any  Curies  specific  calculate  (total  picomoles  of  binding  the  added, (F)  (B/F).  was  inverse  in  the  of  the  represented  the  dissociation  x-intercept  the  uncorrected  numbers  the  Scatchard  the  for  data  Sunahara  5)  to  computer  and F a w z i )  Determination Protein  Bradford linear  for  protein  Perkin-Elmer used  to  minutes  Model  assess after  contained:  Protein  Using  were  124 d o u b l e  of  (Kd),  of  free  a plot). the  plot  the  capacity.  the  bound  observed  of  and  obtained  by  (The subjecting  programme results  by  by hand).  Content  a standard, from 0.1  by t h e the  method  assay  - 0.5  Samples  5 mL B r a d f o r d  100 mg C o o m a s s i e - b r i 1 1 i a n t  were  reagent  blue-G;  50 mL  of  is  mg/mL.  beam s p e c t r o p h o t o m e t e r  concentration.  addition  was  at  ligand  specific  portion  was d e t e r m i n e d  BSA as  the  (Scatchard  (Input/Calc  concentrations  protein the  linear  and by c o n f i r m i n g  of  of  (SB)  total  SB t o y i e l d  slope  measure  analysis  concentration  (1976).  of  constant  plot  binding  represents  for  subtracted  The  where d i s p l a c e m e n t  a negative  slope  were  dpm.  to  normalized  specific  calculation  12  = 2.2x10  was  values  were  employed  averaged.  SB,  as a f u n c t i o n  with  the  the  were  which  the  then  dpms  1 Ci  ligands  give  less  In c a s e s  plot of  to  The  that  NSB t u b e  were  used  graphed  curve-linear The  values  Duplicates  counts)  B/F  Then the  TB t u b e  free  basis the  concentration over  the of  the  point.  on  standards.  activities  content.  the  quench  A  was read 5 which  2 absolute and  filtered  6)  at  ethanol;  a Buchner  Statistical  Analysis  Differences  were  p<0.05  test.  using  100 mL 85% p h o s p h o r i c  using  suction  considered  ANOVA and the  acid;  diluted  to  IL  apparatus.  significant  Newman-Keuls  from  multiple  control range  1  RESULTS  1)  Effect  of  possible  Early  in  our  contamination (Poellinger a blood-borne various in  situ.  at  time  As of  decapitation)  was  assayed  manner.  capacity  or  Kd i n  buffer.  It  can  capable  of  binding  shown  Table  points. state.  II  capacity and  apparent  Diabetes  glycosuria,  was  1/2% or  Table in  in  the  Figure  10 or  whole  out animals  presence  apparent  with  that  from  of  and 1 there  binding  100 mL of  blood  TED  contained  diabetes  of  Figure  injection  of  the  HCLA b i n d i n g  2.  The  binding  both  the  4 day  Kd i s  not  affected  confirmed greater.  60 mg/kg of  by t h e  and  STZ  protein  is  capacity  is  10 day by t h e  presence  time diabetic  of  was  assay,  blood  a specific  I and  the  our  the  the  there  estradiol.  a single  by 50-80% at The  in  perfused  be seen  binding  of  upon t h e  decreased  livers  STZ-induced  The e f f e c t  in  shown  with  flush  for  was made  whether  (taken  estradiol  differences  also  of  bind  As  no s i g n i f i c a n t  Effect  used t o blood  saturable  2)  were  blood  results  may i n t e r f e r e  rat  could  that  determine  whole  which  agent  To  which  volumes  well,  suggestion  artifactual  1983).  component  perfusion  contamination  the  produce  al . ,  component  were  studies  could et  blood  no  any  2 3 Table EFFECT  OF LIVER  I  PERFUSION  VOLUME  The e f f e c t of v a r y i n g t h e l i v e r p e r f u s i o n volume was determined in c i t r a t e i n j e c t e d control animals ( c i t r a t e b u f f e r pH 4 . 5 ) . Rat b l o o d was a s s a y e d f o r t h e p r e s e n c e of any component which was c a p a b l e of b i n d i n g t h e l i g a n d i n a s p e c i f i c and s a t u r a b l e manner. I n c u b a t i o n s were c a r r i e d out as d e s c r i b e d i n t h e methods s e c t i o n . B i n d i n g k i n e t i c s were d e t e r m i n e d by S c a t c h a r d a n a l y s i s . Data were e x p r e s s e d as mean+S.E.M. Numbers i n b r a c k e t s d e n o t e t h e number of animals t e s t e d .  Perfusion Volume  Apparent Kd  Binding  (10" M) 7  Bmax  Kinetics (pmol/mg)  10 mL  1.08+0.34  14.83+3.97  (11)  100 mL  1.41+0.11  17.91+4.82  (8)  Rat  no d e t e c t a b l e  blood  * denotes  no d e t e c t a b l e  displaceable  and  binding* saturable  (2) binding  Fig.  1 Effect  Different buffer the  Methods are  varying  volumes  were  liver.  of  the  liver  (10 mL© through  Incubations  were  Graphs  representative  of  the  vena  carried  shown  the  volume.  -o , 100 m L e a )  perfused  section.  perfusion  are  results  cava  out of  before  according  a single  found.  of  TED  removal to  of  the  experiment  and  Table  II  EFFECT OF STREPTOZOTOCIN-INDUCED DIABETES AND INSULIN REPLACEMENT ON THE MALE RAT HEPATIC HCLA BINDING PROTEIN A d u l t male r a t s were r e n d e r e d d i a b e t i c by a s i n g l e injection of s t r e p t o z o t o c i n (STZ) (60 mg/kg) i n c i t r a t e b u f f e r (pH 4 . 5 ) under e t h e r a n a e s t h e t i c ( c o n t r o l s were i n j e c t e d w i t h v e h i c l e o n l y ) . P r o t a m i n e z i n c i n s u l i n ( P Z I ) was a d m i n i s t e r e d i n a dose of 10 u n i t s / k g s . c . once a d a y , T o r o n t o i n s u l i n ( T o . I ) was g i v e n i n a dose of 15 u n i t s / k g s . c . t w i c e a d a y . I n c u b a t i o n s were c a r r i e d out as d e s c r i b e d i n t h e Methods section. B i n d i n g k i n e t i c s were d e t e r m i n e d by S c a t c h a r d analysis. Data were e x p r e s s e d as mean +_ S . E . M . Numbers i n b r a c k e t s d e n o t e number of a n i m a l s t e s t e d .  Treatment  Apparent Kd  Binding  (10"'M)  Kinetics  Bmax  (pmol/mg)  14.83+3.97  (11)  Control  1.08+0.34  4 day  STZ  0.82+0.18  4.82+0.95*  (8)  4 day  STZ + PZI  0.79+0.19  3.41+0.99*  (5)  STZ +To. I  0.57+0.06  1.41+0.38*  (7)  4 day  10 day  STZ  0.51+0.26  4.37+1.59*  (5)  10 day  STZ + PZI  1.08+0.18  5.72  (5)  * denotes s i g n i f i c a n t (p<0.05) a c c o r d i n g to  1.23*  d i f f e r e n c e compared to c o n t r o l ANOVA and t h e Newman-Keul range  test  2 7 Fig.  2 Effect  of  the  were  HCLA  of  binding  rendered  buffer (A  A)  described  days in  before the  single" experiment found.  in  controls  Animals  of  rat  by a s i n g l e  (60 mg/kg)  anaesthesia; only.  protein  diabetic  streptozotocin ether  streptozotocin-induced  were use.  Methods and a r e  liver. tail  citrate (o  left  for  diabetes  vein  buffer  male  (pH 4 . 5 )  injected  4 (•-  o) or  were  Graphs  representative  carried  shown a r e of  levels rats  injection  o) were  Incubations section.  Adult  on t h e  the  of  under  with 10 out of  results  as a  2 9 3)  Effect  of  insulin  The e f f e c t dose and  of  of  3.  This  restoring  the  4 day  10 day  and  capacity seen  in  control  diabetic  zinc  between  the  insulin the  was t e s t e d in  due t o  a report  (1984)  found  reducing  STZ-diabetic s.c. had  in  some of  twice been  the  binding  seen  in  Table  HCLA  binding  protein  and  its  we were  again  it  dosage  unable  to  The d i a b e t i c  lack  levels 3.  would did  cause  not  in  of  model  was  with  this  With  respect  protein  units/kg once  reduced  a day  HCLA  treatment  the  in  the  of  to  of  al.  15  restoration  was b e i n g  the  seen  et  insulin  that  in  binding  seen  zinc  the  was e f f e c t i v e  a regimen  the  those  reversal  can  the  type  make a d i f f e r e n c e  restore state  the  HCLA  insulin  appear  the  regular  Ortiz-Caro  of  of  4 day  Toronto  dysfunction in  both  glycosuria  the  the  protamine  and F i g u r e  of  points  when g i v e n  in  controlling  time  regular  in  binding  no d i f f e r e n c e s  of  II  unaffected.  in  could  Table  50-80% below  lack  literature.  The  protein  animal  in a  ineffective  were  capacity  whereas  protein  insulin  capacity.  II  the  in  apparent  Kd was  it  thyroid  ineffective.  estrogen  by  if  Toronto  animal  levels  treatment  be seen  The  experiments.  see  the  the  a day,  to  there  binding  that  animals.  10 day  to  can  was e f f e c t i v e  and  STZ-diabetic  insulin  a day  and t h e  Since  of  the  HCLA b i n d i n g  evidenced  rest  in  regimen was  the  depressed  4 day  decrease  once  diabetic  as  animals.  the  of  insulin  treated  zinc  treatment  animals  state  for  s.c.  levels  remained  Protamine  used  protamine  10 u n i t s / k g  Figure  replacement  of  as  binding  controlled  as  be  3 0 Fig.  3 Effect  binding Adult  male  of  Toronto  regular  twice  Incubations section.  4 (•  only.  insulin for  were  Graphs  representative  shown the  are  diabetic  citrate  controls  once  zinc  insulin  A ) days  before  a dose  of  (v  v)  before  use.  results  was  Animals  a single found.  vein  buffer  a day.  as d e s c r i b e d  tail  (pH  CD) were  fp  in  of  rats.  in  given  out  HCLA  (60 mg/kg)  10 ( A  4 days  hepatic  a single  Protamine  was  carried  of  the  by  s.c.  •) or  a day  of  diabetic  anaesthesia;  10 u n i t s / k g  for  s.c.  rendered  buffer  treated  level  streptozotocin-induced  were  ether  of  on t h e  streptozotocin  with  a dose  in  rats  under  injected in  insulin  protein  injection 4.5)  of  in  15  the  experiment  given  were use. units/kg  Methods and  are  3 2 evidenced  4)  by the  Effect  of  vein  saline or  in  under  corn  The  that  there  capacity  citrate  was  is  injections  buffer  s.c.  injected can  at  injection (pH=4.5)  aneasthesia)  results  or  glycosuria.  subcutaneous  injected  oil  day.  of  ether  was  of  subcutaneous  The e f f e c t determined  lack  control  a volume  animals. of  0.3  at  a volume  be seen  in  Table  Kd between  in  either  injected  the  or  vehicles  injected  s.c.  no change  of  (via  0.5  III.  a  tail  Normal  mL once  of  was  It  apparent  a day,  mL once is  a  apparent  binding  non-injected  control  ani m a l s .  5)  Effect  of  a)  Testosterone Due t o  diabetic on t h e  the  male  diabetic of  the  that  plasma  unable  to  seen  in  protein  can  this  of  be  dose,  the  diabetic  in  the  of  in  which  is  animals.  in  enanthate and  (Sunahara,  binding  the  m a l e , we  replacement  IV  in  testosterone  sufficient  The  seen  gonadectomized  Table  levels,  animal.  of  testosterone  decreased  control  effect  testosterone  seen  testosterone  the  serum t e s t o s t e r o n e  restorative  effect  restore  STZ-induced  in  The e f f e c t  1 mg/kg  normal  that  decrease  and t h e  male.  observed  replacement  enanthate  HCLA b i n d i n g  investigated  dose  hormone  Figure to  Kd r e m a i n s  the  The d i a b e t i c  at  a  4.  It  is  restore  1984)  capacity  the  is  seen same  in as  animals  the  3 3 Table EFFECT  III  OF SALINE AND CORN OIL S . C . IN CONTROL ANIMALS  INJECTIONS  A d u l t male r a t s were i n j e c t e d w i t h 0 . 3 ml c i t r a t e b u f f e r (pH 4 . 5 ) under e t h e r a n e a s t h e t i c . Normal s a l i n e was i n j e c t e d s . c . at a volume of 0 . 3 ml once a d a y . Corn o i l was i n j e c t e d a t a volume o f 0 . 5 ml once a d a y . I n c u b a t i o n s were c a r r i e d out as d e s c r i b e d i n t h e Methods s e c t i o n . Binding k i n e t i c s were d e t e r m i n e d by S c a t c h a r d a n a l y s i s . Data were e x p r e s s e d as mean _+ S . E . M . Numbers i n b r a c k e t s d e n o t e number of a n i m a l s t e s t e d .  Treatment  Apparent Kd  Saline Corn  oil  Control  s.c. s.c.  (10  Binding )  Kinetics  Bmax  (pmol/mg)  1.01+0.01  14.71+1.53  (4)  0.90+0.01  12.68+2.83  (4)  1.08+0.34  14.83+3.97  (11)  treated at  the  b)  with time  of  the  there  diabetic  are  IV and  Figure  thyroid that  the  animal but  to  signs  diabetic  c)  of  unable  glycosuria  for  The  were  restore  restore  out  of  the  (Tahiliana,  the  with  at  in  decreased  from t h e  in  1983).  triiodothyronine  control  the  time  of  in  Table  restoring It  can  the  be  the seen  diabetic  binding  capacity  values.  The  triiodothyronine  replacement 5.  tested  killing.  growth  with  male  of  the  these  levels release  decreased  growth  of  1976)  use per  of  pattern  binding  from  can  known  i)  day s . c .  of  regimens vi a  ii)  (done  and t o  respectively)  capacity  seen  injection  regimens  hormone  be  blood  replacement  minipumps;  treatment growth  hormone  two t r e a t m e n t  hormone:  v i a a seven t i m e s Neither  of  Calculating  and M a r t i n ,  with  infusion  absolute  natural  effective  be seen  of  hormone  carried  schedule.  is  treated  (Tannenbaum  replacement  dosage  restore  IV and F i g u r e  continuous  ng/kg  of  glycosuria  effect  Table  30  can  levels  replacement  of  unaltered  animals  the  hormone  administering  status  to  thyroid  of  a dose  This  normal  Growth  levels  4.  Kd was  postive  at  in  we i n v e s t i g a t e d  The e f f e c t  replacement  is  the  alterations  animal,  triiodothyronine  the  showed  killing.  triiodothyronine.  in  still  3,3,5-triiodothyronine Since  in  testosterone  the  to  duplicate  were HCLA  able  to  binding  3 5 Table EFFECT  IV  OF HORMONE REPLACEMENT ON THE RAT HEPATIC BINDING PROTEIN IN 4-DAY DIABETIC ANIMALS  HCLA  A d u l t male r a t s were r e n d e r e d d i a b e t i c by a s i n g l e injection of s t r e p t o z o t o c i n • ( S T Z ) (60 mg/kg) i n c i t r a t e b u f f e r (pH 4 . 5 ) under e t h e r a n a e s t h e s i a . T e s t o s t e r o n e e n a n t h a t e was g i v e n i n a dose of 1 mg/kg i n c o r n o i l once a d a y . T r i i o d o t h y r o n i n e was g i v e n i n a dose of 30 ug/day s . c . i n normal s a l i n e . The a n i m a l s which had o s m o t i c minipumps i m p l a n t e d were g i v e n a dose of o v i n e growth hormone ( 1 . 5 I . U . / m g ) at 0.02 u n i t s / h o u r f o r f o u r d a y s . The dose of o v i n e growth hormone g i v e n s . c . was 30 u g / i n j e c t i o n seven t i m e s a d a y . I n c u b a t i o n s were c a r r i e d out as d e s c r i b e d i n t h e Methods s e c t i o n . B i n d i n g k i n e t i c s were d e t e r m i n e d by Scatchard a n a l y s i s . Data were e x p r e s s e d as mean +_ S . E . M . Numbers i n b r a c k e t s d e n o t e number of a n i m a l s t e s t e d .  Treatment  Apparent Kd  Binding  (10~'M)  Kinetics  Bmax  (pmol/mg)  14.83+3.97  (11)  Control  1.08+0.34  4 day  0.82+0.18  4.82+0.95*  (8)  0.56+0.10  3.65+0.82*  (5)  4 day STZ +t r i i o d o t h y r o n i ne  0.40+0.10  1.83+0.40*  (5)  4 day STZ +growth hormone  (minipump)  0.56^0.11  1.81+0.22*  (5)  4 day STZ +growth hormone  (s.c.)  0.52+0.06  1.54+0.28*  (7)  STZ  4 day STZ +testosterone  enanthate  * denotes s i g n i f i c a n t (p<0.05) a c c o r d i n g to  d i f f e r e n c e compared t o c o n t r o l ANOVA and t h e Newman-Keuls range  test  3 6 Fig.  4 Effect  of  testosterone  replacement  in  4 day  streptozotocin-induced  animals  on t h e  level  of  liver.  Adult  vein  injection  buffer were  ug/kg  of  (pH 4 . 5 )  injected  testosterone once  male  a day in  section.  ether  at  Animals  a dose  of  saline  s.c.  were c a r r i e d  Graphs of  out  shown are the  once  of  results  a day  a single  rat  (o  were t r e a t e d  at (A  tail  citrate  controls  as d e s c r i b e d  found.  in  1 mg/kg  • ) , or t r i i o d o t h y r o n i n e  of  by a s i n g l e  (60 mg/kg)  anaesthesia;  only.  diabetic  protein  were made d i a b e t i c  buffer  enanthate  representative  HCLA b i n d i n g  streptozotocin  with  normal  Incubations  the  rats  under  (b  and 3 ,3 , 5 - t r i i o d o t h y r o n i n e  in  corn  a dose  of  o) with oil 30  .—-A). in  the  experiment  Methods and  are  s.c.  0  2  Specific  4  Binding  6  B  (ptnol/mg)  10  3 8 Fig . 5 Effect  of  replacement  ovine  on the  growth  hepatic  streptozotocin-induced rendered  diabetic  streptozotocin ether  only.  saline  and  a dose  0.02  of for  the  described single found.  in  the  experiment  or  seven use.  Adult  were  was  osmotic the  injected  s.c.  a day.  section.  male  rats  in  minipump rat's  back  (A  A)  Graphs  shown the  with (pH (•  at  out are  8.5) •)  and  were  carried  of  under  basic  were  were  of  (pH 4 . 5 )  Animals  representative  4 day  injected  dissolved  of  in  injection  buffer  (o  Incubations  are  protein  vein  citrate  times  Methods and  tail  portion  units/hr,  before  rats.  by e i t h e r  rostral  30 u g / i n j e c t i o n 4 days  hormone  administered in  in  controls  Growth  implanted of  diabetic  (60 mg/kg)  I.U./mg)  (1.5  HCLA b i n d i n g  by a s i n g l e  anaesthesia;  buffer  hormone  given  a dose  treated as of  results  a  0  2  Specific  4  Binding  6  8  (pmol/mg)  4 protein  in  unaltered animals  the  diabetic  from t h e  tested  rat.  value  seen  positive  for  6)  Effect  of  intravenous  a)  Vena cava  cannulation  The e f f e c t administration can  be  seen  duplicate (i.e. of  to  the  of  in  the get  vena  marked  gave  we o b s e r v e d ;  blood  raised  over  the  potential  anaesthetic  and  a control  halothane  anaesthetic. There  were  values but  The  obtained  these  obtained  from c o n t r o l  manipulated; cannulated values. precluded  were  the  groups  The  low  seen  There  of  the  was  differences  in  halothane  number  of  manipulation  or  in  ether than  not  same  in  used t o of  to  as  was  an  compare  the  Table  binding  different  animals  control  concern  halothane done  the  had  not  from t h e  be seen  animals  and were  protein  the  surgical  lower  were t h e  blood  treatment  of  significantly which  closely  restoration  capacity  as t h e can  the  no  unaltered  was  animal  more  HCLA b i n d i n g  binding  this  subsequent  to in  and t h i s  ether  of  and t h e  undertaken  experiment  either  the  administration  STZ-diabetic  hepatotoxiciy  Kd v a l u e s  statistical  the  level  low  versus  results  with  values  was  remained  very  no a p p a r e n t  and  cannulation  levels  Because  of  animals,  peaks).  Kd  this  control  capacity  lowest the  was  hormone  physiologic  value.  effects  of  growth  This  binding  the  Kd v a l u e  hormone t o  V.  normal  in  the  glycosuria.  cava  growth  Tabel  decreased  actually that  of  Again,  0  capacity  anaesthesia, the  been all  V.  values  surgically three  from t h e obtain figures.  control data Larger  Kd  4 1 Table V EFFECT OF VENA CAVA CANNULATION ON THE THE RAT HEPATIC HCLA BINDING PROTEIN A d u l t male r a t s were c a n n u l a t e d as d e s c r i b e d i n t h e Methods s e c t i o n under e i t h e r e t h e r or h a l o t h a n e a n a e s t h e s i a . One group was r e n d e r e d d i a b e t i c by a s i n g l e i n j e c t i o n of s t r e p t o z o t o c i n (STZ) (60 mg/kg) i n c i t r a t e b u f f e r (pH 4 . 5 ) w h i l e under t h e o p e r a t i v e a n a e s t h e t i c ( c o n t r o l s were i n j e c t e d w i t h b u f f e r o n l y ) . Ovine growth hormone ( 1 . 5 I . U . / m g ) was g i v e n i n a dose of 30 u g / i n j e c t i o n s . c . seven time a day. B i n d i n g k i n e t i c s were d e t e r m i n e d by S c a t c h a r d a n a l y s i s . Numbers i n b r a c k e t s d e n o t e t h e number of a n i m a l s t e s t e d . Data were p r e s e n t e d as the mean +_ S . E . M . I n c u b a t i o n s were c a r r i e d out as d e s c r i b e d i n t h e Methods section.  C o n d i t i o n s of C a n n u l a t i on  Apparent Kd  ether  anaesthetic  Binding  ( 10 ' ' M)  Kinetics Bmax  (pmol/mg)  0.53+0.17  2.85+1.67  (3)  anaesthetic  0.59+0.13  4.25+2.44  (3)  halothane anaesthetic +STZ t g r o w t h hormone  0.46+0.20  0.51+0.19  (2)  halothane  numbers of  the  of  animals  rat  to  the  cannula  was  unusable,  cannula b)  out,  vein  The hormone  shown  with  control the  showed  doses  growth with  treated  protein,  showed hormone  the  insulin  conscious  and  All  Figure  untreated  (i.e.  was  were  evident  only  animals  binding  from  inside  10-15  zinc  group  insulin in  to  compared  this  which  to  that  animals at  also the  to to 5  with  level,  i.e.  treated  testosterone given, i.v.  animal  in  growth  seen w i t h  the  injected  except the  with  an  hormone  were  glycosuria  vehicle  injected  (injected  below  STZ-diabetic  injected  showed  in  addition  similar  and t h e STZ  growth  the  pmol/mg p r o t e i n  controls  HCLA  This  being  showed  the  uninjected)  in  capacity  50% d e c r e a s e the  of  Kd v a l u e s .  also  low.  treatment  capacity  the  the  growth  binding  in  treatment  6.  it  animal  of  in  as d i d  were  administration  These  a decrease  All  the  in  where  plugging  numbers  the  previously,  alone  clots  final  which  The STZ  end down t o  blood  the  i.e.  and p r o t a m i n e  with  injection  VI  vein.  A second  saline.  pulling  to  a further  described  hormone  its  capacity  controls,  saline)  enanthate  by  the  50% d e c r e a s e  protein.  diabetics.  as d e m o n s t r a t e d  in  animals  tail  pmol/mg  2 pmol/mg  Table  binding  approximately  to  intolerance  no d i f f e r e n c e s  injected  non-injected  the  vein  compared  in  in  tail  in  but  p r o b l e m of  a decrease  decrease  saline  of  the  animal,  of  protein  controls,  because  administration  showed  binding  chewing  the  effect is  used,  cannula,  and t h e  inside  Tail  groups  the  were  time  those of  Table  VI  EFFECT OF HORMONE REPLACEMENT BY TAIL VEIN INJECTION IN CONSCIOUS 4-DAY DIABETIC ANIMALS ON THE RAT HEPATIC HCLA BINDING PROTEIN A d u l t male r a t s were r e n d e r e d d i a b e t i c by a s i n g l e injection of s t r e p t o z o t o c i n (STZ) (60 mg/kg) i n c i t r a t e b u f f e r (pH 4 . 5 ) under e t h e r a n a e s t h e s i a ( c o n t r o l s were i n j e c t e d w i t h buffer only). O v i n e growth hormone ( 1 . 5 I.U./mg) was i n j e c t e d i . v . at a dose of 30 u g / i n j e c t i o n f o u r t i m e s a day i n normal s a l i n e ( v e h i c l e i n j e c t e d c o n t r o l s and v e h i c l e i n j e c t e d d i a b e t i c c o n t r o l s were i n j e c t e d w i t h s a l i n e o n l y ) . T e s t o s t e r o n e e n a n t h a t e was g i v e n at a dose of 1 mg/kg s . c . i n c o r n o i l once a d a y . P r o t a m i n e z i n c i n s u l i n ( P Z I ) was g i v e n i n a dose of 10 u n i t s / k g s . c . o n c e a d a y . I n c u b a t i o n s were c a r r i e d out as d e s c r i b e d i n t h e Methods s e c t i o n . B i n d i n g k i n e t i c s were d e t e r m i n e d by S c a t c h a r d a n a l y s i s . Data were e x p r e s s e d as mean _+ S . E . M . Numbers i n b r a c k e t s d e n o t e number of a n i m a l s t e s t e d .  Treatment  Apparent Kd  (10" M) 7  1.08+0.34  Control Vehicle Control  Binding  injected  Kinetics  Bmax  (pmol/mg)  14.83+3.97  (11)  0.66+0.11  4.05+0.47*  (4)  Vehicle injected 4 day STZ  0.36+0.03  2.07+0.37**  (4)  4 day STZ +growth hormone  0.43+0.04  2.25+0.34**  (4)  4 day STZ +growth hormone +testosterone enanthate + PZI  0.54+0.18  2.37+0.37**  (4)  * d e n o t e s a s i g n i f i c a n t d i f f e r e n c e from c o n t r o l (p<0.05) a c c o r d i n g t o ANOVA and t h e Newman-Keuls range t e s t ** d e n o t e s a s i g n i f i c a n t d i f f e r e n c e from c o n t r o l and v e h i c l e i n j e c t e d c o n t r o l ( p < 0 . 0 5 ) a c c o r d i n g t o ANOVA and t h e NewmanKeul s range t e s t  4 4 Fig.  6 Effect  of  tail  vein  streptozotocin-induced binding single  protein. tail  citrate  buffer  (•———•) and vein  vein  one  were  with  of  Another  group  hormone  dissolved  use  (A  growth  Adult  injection  of  alone  (•  a day  in  in  last basic  enanthate plus  s.c.  Graphs  representative  of  a tail of  (pH 8 . 5 )  a day at  in  zinc  vein  of  with  insulin  at  before  out  as d e s c r i b e d a single found.  use.  growth  a dose  of  for  4 days  before  ,  of  30  with  30  plus  1 mg/kg  carried  results  before  a dose  vein  tail  at  at  4 days  the  a  saline  for  of  in  was t r e a t e d  a day  shown are  o)  (D—  4 days  saline  in  controls  injected  for  by a  diabetics  a tail  a dose  protamine  once  were  group  HCLA  (60 mg/kg)  was t r e a t e d  a day  The  hepatic  Control  •) were  animals  4 day  anaesthesia; only.  4 times  testosterone  section.  buffer  (pH 8 . 5 )  4 times  Incubations  ether  4 times  diabetic  on the  streptozotocin  basic  hormone  units/kg  with  conscious  were made d i a b e t i c  in  A).  a day,  of  in  rats  rats  under  diabetics  ug/injection  once  male  (pH 4 . 5 )  saline  ug/injection  diabetic  injected  group  injections  in  corn  a dose use in  of  (v  the  experiment  oil  s.c.  10 v)  .  Methods and  are  4  0  2  Specific  4  Binding  6  8  (pmol/mg)  5  killing. study seen  Thus,  were in  none  able  the  to  capacities  vehicle  injected hormone  significant, between That the  the  is,  7)  of  Other  the  species  be seen  species  examined  showed  protein  in  liver  Stability It  seen  may be due t o during results  the  still  is  a  and  the  and  animals  diabetic  of  the  were  difference  causing  also  animals  an e f f e c t  handling  occuring  examined  protein. in  Table  The VII.  any e v i d e n c e  on  and  in  parallel.  for  the  presence  results  of  None of  the  of  the  HCLA  these other  binding  cytosol.  suggested  between  the  differences  that  in  be seen  Table  in  cytosols  the  control  period.  STZ-diabetic  controls,  treated  stress  were  incubation  can  between  studies  had been  capacity  is  is  capacity  hormone t r e a t e d ,  and t h e  the  effect  HCLA b i n d i n g  their  difference  + insulin  protocol  this  binding  injected  there  through  in  in  examined  other  can  The  growth  animals  this  diabetic  species  experiments  8)  that  involved  decrease  vehicle  + testosterone  probably  hepatic  the  STZ c o n t r o l s ,  non-diabetic  animal,  treatments  animal.  the  indicating  Several the  reverse  even though  injection,  the  STZ-diabetic  binding  growth  of  the  differences  in  and S T Z - d i a b e t i c stability  This VIII.  demonstrated  of  the  was examined Both  the  binding animal protein  and  the  control  and  a stability  of  binding  of  capacity this  case  capacity times the  and Kd up t o 3  was e v i d e n t  higher  two  hours).  than  groups  the The  longest observed  (i.e.  same.  point  difference  controls  STZ-diabetics)  were t h e  time  being  and the  measured in  the  (in  binding  approximately Kd v a l u e s  3  between  4 Table OTHER  MODELS  8  VII  EXAMINED FOR THE PRESENCE HCLA BINDING PROTEIN  OF THE  Mouse and g u i n e a p i g l i v e r was a s s a y e d f o r t h e HCLA b i n d i n g p r o t e i n i n t h e same manner as r a t l i v e r . Binding k i n e t i c s were d e t e r m i n e d by S c a t c h a r d a n a l y s i s . Numbers i n b r a c k e t s d e n o t e t h e number of a n i m a l s t e s t e d . I n c u b a t i o n s were c a r r i e d out as d e s c r i b e d i n t h e Methods s e c t i o n .  Animal  model  Apparent Kd  C57BL/6  mouse  DBA/2 mouse guinea  pig  1i v e r  liver liver  Binding  (10 ' M ) 1  Kinetics  Bmax  (pmol/mg)  no d e t e c t a b l e b i n d i n g * (4 p o o l s of 3) no d e t e c t a b l e b i n d i n g * (4 p o o l s of 3) no d e t e c t a b l e  binding*  (4)  * denotes  no d e t e c t a b l e  displacable  and  saturable  binding  Table  VIII  STABILITY STUDY OF THE HCLA BINDING PROTEIN IN CONTROL AND 4-DAY DIABETIC ANIMALS A d u l t male r a t s were made d i a b e t i c by a s i n g l e i n j e c t i o n of s t r e p t o z o t o c i n (60 mg/kg) i n c i t r a t e b u f f e r (pH 4 . 5 ) under e t h e r a n e a s t h e t i c ( c o n t r o l s were i n j e c t e d w i t h b u f f e r o n l y ) . C y t o s o l was i n c u b a t e d i m m e d i a t e l y (0 h r . ) , or l e f t sitting on i c e f o r 1 hr b e f o r e u s e , or l e f t s i t t i n g on i c e 3 h r . b e f o r e u s e . I n c u b a t i o n s were c a r r i e d out as d e s c r i b e d i n t h e Methods s e c t i o n . B i n d i n g k i n e t i c s were d e t e r m i n e d by Scatchard a n a l y s i s . Data were e x p r e s s e d as mean+_S.E.M. Numbers i n b r a c k e t s d e n o t e number of a n i m a l s t e s t e d .  Treatment  Apparent Kd  Cont r o l 0  hr.  1  hr.  3  hr.  4 day  diabetic  Binding  (10" M) 7  Kinetics  Bmax  (pmol/mg)  0.72+0.01  12.59+0.66  (2)  0.70+0.01  13.29+0.68  (2)  0.89+0.02  12.96+0.58  (2)  0.47+0.02  2.78+1.56  (2)  0  hr.  0.60+0.01  3.98+0.15  (2)  1  hr.  0.68+0.01  3.47+0.46  (2)  3  hr.  5 0 DISCUSSION  1)  Binding The  binding male  results  liver  protein)  are  et  saturable  volume,  was  liver  cytosol and  steroids was  blood  borne  lack  the and sex  not  artifactual  rat  2)  Effect  can  be  could  binding  in  et  binding  to  also  detect  al.,  whole  non-specific had  is  globulin  interfering  results  assay  with  which  been  from  to  the  in  proteins which  can  1982).  might  bovine  data.  rat of such  as  also  bind  Since  there  the  used,  perfusion  were  assay lead  demonstrated  binding  specific,  a component  blood  The  system  liver  transport  Siiteri  that  be d e t e c t e d  protein  plasma  detected  the  authors  1983).  generated  varying  binding  1974;  the  of  pmol/mg  by o t h e r  indicate  contaminants  (1983) who f a i l e d and  I)  the  protein  Finlayson,  in  of  the  hormone  no b i n d i n g  of  reported  place  HCLA b i n d i n g  (Anderson,  there  The  no e f f e c t  binding  for  capacity=14.83  1982;  plots  no s p e c i f i c  blood,  albumin  taking  Scatchard  whole  those  (see A p p e n d i x is  experiments  binding  1980; W a r r e n ,  binding  and  to  these  HCLA e s t r o g e n  7  curves  there  the  similar  reliable  Since  of  in  (Kd=10~ M,  al . ,  saturation  and  obtained  kinetics  rat  (Eagon  kinetics  by  no  system.  to Finlayson  serum  albumin  plasma.  of  STZ-induced  STZ-induced  diabetes  diabetes caused  a decrease  of  50-8 0% i n  the  5 1 binding and  capacity  10 day  levels  of  indicate and As  is  the that  The  appears  to  protein  per  its  binding  to  Appendix  et is  in  se as  (but  as to  Table  VIII  as t h e  at  reduced  its  decrease  be t h e  al. , not  hepatic  al.,  1983; Warren a generalized  diabetic  state;  proteins  are  affected  proteins  diabetic  rats  increased. occur  and t h e  seen  the  time  could  the  (i.e.  effect is  (1974)  are  1983)  STZ-induced  of  in in  groups  also  show  the the  diabetic  and  of  not  observed in  the  (Reinke  that in  the  other  levels  of  the  spontaneously rats.  a  1980;  proteins  decreased  parallel  does  and C o o k ,  be  there  concentration  by o t h e r  the  HCLA  and H e r r a r a  Alterations in  in  of  as  This  al.,  both  there  proteins).  protein  indicating  for  It  a direct  action  depression  some p r o t e i n s  diabetic  stability  capacity).  early  and  over  seen  points,  period.  that  the  The  time  binding,  control  1979 ; P a s t  et  noted.  occurs  STZ t r e a t m e n t ,  Observations et  control  be due t o  as Montoya in  this  the 4  time  capacity  in  decrease  hepatic  both  indicate  diabetogenic  animal. Stohs  the  could  in  case,  increase  1978;  its  both  be g e n e r a t e d  alteration the  at  Kd was  at  saturable  stable  levels  the  could  whether  to  both  of  the  within  plots  a result as  in  binding  specific,  results  STZ-diabetic al. ,  I,  protein  equal  regeneration  Scatchard  protein  an o v e r a l l  there  in  unrelated  seem t o  Peng  reduction  be no a p p a r e n t  generalized  et  in  studied  questioned  STZ,  the  remaining  course  No c h a n g e being  subject  reliable  HCLA b i n d i n g  points.  demonstrated  groups.  by  the  HCLA p r o t e i n ,  be seen  animals that  time  not  can  of  More  specifically,  with  Warren  using  (1982)  regard  diabetic  change  binding  the  upon t h e  application  computer  program  control pools were to  examine  to  see  could  if  the  These  any  lack  be due t o  of  the  assay  (Finlayson,  that  of  Warren  agree  towards  reduced  diabetic  animal  state  i.e.  indicating and of  not any  observed that the  number  absolute available  of  all  to  the  effect  Kd i n This  that  binding  amount  of  reduction  on t h e  by W a r r e n .  probably  the  of  is  the  ligand  by  Scatchard  animals  in  using  the  due t o  diabetic  of  the  STZ-treatment. was  in  The  the  Kd  altered  in  the  diabetics  produced, cytosol  or in  indicates  protein, the  lack  also  in  on t h e  the  state  change  available  the  state  and  disease  is  diabetic  this  trend  protein of  the  the  report  a general  etiology  rats  analysis  observed  this  we  Warren  induced)  protein in  obtained  chemically  of  from  tissue  or  being  sites  noted  B.B.  HCLA b i n d i n g the  the  lack  is  was  diabetic  the  but  fitting  Unfortunately,  However,  there  spontaneous  a nonspecific effect  of  in  analysis,  done w i t h  those  noted  that  (regardless  the  were  variation  1983).  capacity  either that  in  as marked a  curve  seen w i t h  individual  examining  Scatchard  spontaneously  levels  see  animals.  to  protein,  and  a 60% d e c r e a s e  of  change  protein  system  using  results  HCLA b i n d i n g  not  non-linear  number  obtain  system  did  experiments  and compare the  rats  capacity of  and on a s m a l l to  B.B.  HCLA b i n d i n g  assay  (NONLIN UBC)  values.  unable  the  a similar  spontaneously in  to  is  the  amount  a functional  form.  5 3 3)  Hormone  a)  Insulin  replacement  Insulin once  per  levels which  day,  which had  diabetes  the  the  in  In c o n t r a s t ,  1982). not  the  reversing  time  with  of  there  does  This  is  not  in  normal insulin  demostrates factor  This  that in  the  appear  contrast  to  is  insulin  and e s t r o g e n  Hif,  b)  1978 ;  Lui  previously  levels adult the  treatment  al. ,  capacity  at  both  of  10  days).  metabolism  (Warren,  levels  per  se  are  protein  be an i n t e r p l a y estrogen  levels,  between  receptor  between  levels  insulin,  (Shafie  and  1981).  Testosterone The dose  had  et  were  ( 4 and  receptor  at  of  effects  binding  an i n t e r r e l a t i o n s h i p  day)  the  drug/steroid  the  insulin  and  HCLA b i n d i n g  to  per  was e v i d e n t  insulin  system where t h e r e receptors,  in  zinc  glycosuria,  metabolism,  investigated of  4 days  a controlling  two.  points  reverse  decrease  protein.  twice  controlling  shown t o  the  restoration  This  and t h a t  been  in  protamine  insulin  drug/steroid  HCLA b i n d i n g  treatment  with  regular  were e f f e c t i v e  on h e p a t i c  was e v i d e n t  (either  or T o r o n t o  previously  ineffective of  replacement  of  testosterone  been  shown t o  testosterone  castrate  levels  of  of  male the  and  enanthate be a b l e  normalize  (Sunahara,  HCLA  binding  1984)  to  used  in  this  restore  circulating  AHH a c t i v i t y as w e l l  protein  in  as  the  study  in  the  restoring gonadectomized  5 4 adult  male  evidence  (Finlayson,  of  restoration  with  this  that  a longer  treatment  restoration was  after  time the  1983).  in  of  the  the  treatment  course  was  testosterone  not  it  course  is in  may have  done.  however,  animal.  the  It as  levels  could  it  be  the  gonadectomized  occured  was  no  protein  necessary  However,  treatment  was,  HCLA b i n d i n g  diabetic  seen by F i n l a y s o n  10 d a y s ;  There  group  sooner  but  should  be noted  initiated  at  the  a that  same t i m e  as  STZ.  c)  3,3,5-triiodothyronine The dose  previously  of  been  triiodothyronine shown t o  the  diabetic  animal  the  diabetic  state  of  this  indicating  role  in  they  do work  the  pituitary  d)  correct  hormone between  there to  hormones  the  content  al.,  status  was no  the are  HCLA b i n d i n g  some of  hormone et  the  of  not  in  in  binding  playing  protein,  the  in  effect  HCLA  abnormalities  seen  has  alterations  There  levels  thyroid  study  thyroid  other  1983).  on t h e  of  any  this  a  though  in  diabetic  1984).  hormone  Growth  leads  to  (Ortiz-Caro  difference  that  in  normal  cause  (Tahiliani,  regulation  growth  Growth  female  not  hormone t r e a t m e n t  protein,  animal  but  restore  used  is  stable,  secretion the  male  a continuous constantly  patterns  and f e m a l e release detectable  show  a marked  animal. of  In  growth  levels  in  the  hormone the  which  circulation. ultradiene are  rapidly  peak. hours  cleared growth  This in  during  the  continuously  rat. the  The  growth  hormone were  levels  of  growth  release which  in  is  experiments protein  to  control  difference  between  is  not  rather  suprising, similar  in  since  a s.c.  there  is  dosing  in  a similar  failed  to  restore  feminizing  the  related  to  the  the  the  levels  in  drug/steroid  or  that  the  per  that  these  binding there  treatment  probably such  was  acts as  seen  in  the  the  in  the  treatments  HCLA  growth  changes  into  probably  these  binding  hormone  seen  no  regimens  diffusion  are  metabolism  with  day)  of  HCLA  two t r e a t m e n t s  theory  involved  the  constant  That  out  Neither  preparation  pattern.  and  male  times  and v a l l e y s  which  are  normal  two  injection  decreased  negate  factor  of  a slow  peaks  of  seen  circulating  which  male.  3.5  that  carried  retrospect,  a depot  Thus,  not  In  twice  seven  elevation  effects  result  function  diabetic  the  is,  the  injections  levels.  to  mimic  secretory  hormone  absolute  which  every  (Tannenbaum  ( v i a minipumps)  system.  does  restore  the  and no marked  the  to  the in  female  two e x p e r i m e n t s  and t o  the  protein,  the  an  no  next  approximately  about  in  (peaks)  the  is  s.c.  manner  until  cycle  That  circulatory  occurs  follows  leaving  male's  minipump. blood,  circulation,  growth  done  resulted  levels  of  hormone  feminized  high  level  first  (via  secretion  The  diabetics,  pattern  to  the  (troughs)  cycle  present  1976).  decreased  leads  hormone  male in  though,  from t h e  rhythmic  a peak  Martin,  male  rhythm which  detectable  is  In t h e  in  (Wilson,  is  liver 1969;  1970;  Mode  presence causes  is  This  normal  would  male for  clearance  of  trough)  growth  as  from t h e  the  growth  are  the  levels  of  and c l e a r i n g  of  levels  al. ,  decreased effect  is  in not  in  is the  (the  physiological of  probably  (Jansson  et  growth  1984).  seen  while  The m a i n t e n a n c e is  (Virgo,  peak)  circulation  various  Its  absence  pattern  (the  inhibiting  et  are  its  1985).  metabolism  hormone  circulation  (Hall  and  ultradiene  from t h e  testosterone capable  Virgo,  and d e v e l o p m e n t ,  hormone  in  pituitary  inhibitory  drug/steroid  be m a i n t a i n e d .  hormone  testosterone  of  and  feminization  masculinization  to  which  with  some growth  growth  circulating  1984)  Vockentanz  explain  normal  allows  parameters  upon  1981;  associated  necessary  of  al . ,  masculinization  1985). the  et  a low  level  dependent al . ,  hormone  release  Since  diabetes seen,  this  leading  to  femi ni z a t i o n . Since duplicate  the the  intravenous  s.c.  injection  normal  secretory  injections  to  effectiveness  of  injections  continuous  normal been  or  growth  ineffectiveness  of  binding  levels  protein  lack  were  added  of to  effect the  the  intavenous  et  growth  in  al.,  again  remained treatment  we d e c i d e d peaks.  over  infusion  the  male  1985).  hormone t o  was  hormone d i d  plasma  injections  hormone p a t t e r n s (Clark  growth  pattern,  obtain  intravenous  demonstrated  This  of  seen  either in  rat  try  The s.c.  recently  The  restore  the  (Tables  to  to  duplicating has  HCLA  V and  when t e s t s o t e r o n e regimen  not  restore  and  VI). insulin  a more  normal  5 7 physiology,  e.g.  baseline  growth  of  injecting the  four  of  per  of  seen w i t h  of  This HCLA  hepatic  four  to  obtain  after  day,  pattern.  restoration  normalization been  hormone  times  ultradiene  lack  testosterone  the  not  drug  may have  and  injections  seven  had an e f f e c t  on  protein  day  We were  physiological  steroid  per  normal  injection.  the  binding  a more  levels  the  although  metabolism  (Skett  of  and  has  Young,  1982). These Animals  studies  which  controls  had been  undergoing  showed m a r k e d l y compared  to  6).  level  The  injected  (Table  in  surgery  and  was  reported surgery days. times to the  animals  and  stress  lower  day  et  the  that  of  al. ,  the  the  these  vein  surgery  to  tail  and  the  levels  vein  be u n d e r g o i n g this  subsequent  It  protein  tail  vein  binding  animals control (i.e.  caused has  a decrease  been  stress for  of  several  injections stress  would  Fig.  non-  manipulations  diabetes.  presumably  HCLA  injection)  day  V and V I ,  reduced  1975 ) t h a t  undergoing  appeared  HCLA b i n d i n g  diabetic  50% below  per  50% from t h e  of  injected  tail  4 times  non-diabetic  levels  that  non-diabetic  (Tables  serum t e s t o s t e r o n e  handling; of  The  appeared  to  the  approximately  vein  conscious  of  controls in  complication.  and t h o s e  injections  levels  a further  (Nakashima  per  pain  was  additive  The  vein  decrease  tail  thus  can  tail  controls.  VI) was It  which  of  by a f u r t h e r  cannulated  non-manipulated  the  value.  marked  reduced  controls  manipulated protein  were  four  probably  be s i m i l a r  intolerance  of  the  due to  5 8 cannula.  This  decreased  levels  and  the  lowering of  further  Another  with  interaction  which  (Higuchi  al.,  et  decreased pathway  in  to  1981) rats  (i.e. react  hormone factor  et  of  for  mice might  the  These  guinea  variation Since  by  al . ,  in  also  be  1 977 ) w h i c h  the  (Terry  and  secretion  the  this  evidence  Martin,  release).  would  in  of  is  in  cause the  are  seen.  and t h e r e  secretion  involvement  diabetics.  be v i a an  ability  involved  to  controls,  release  levels  the  stress  act  the  corticotropin  would  is  could  androgen  inhibiting  seen  where be an  the  binding  presence  was of  particular  pigs)  genetics  protein  by  in  to  However, of  suggest  effect  growth that  this  seen.  species  protein. mice,  and  lead  depressed  levels  a reduction  hormone  protein  prolactin  mechanism  major  No d e t e c t a b l e examined  Since  could  already  adrenal  elevated  stress  not  Other  to  catecholamines to  the  testosterone  growth  (Terry is  in  1984).  an a - a d r e n e r g i c of  HCLA b i n d i n g  prolactin  leads  are  testosterone  testosterone  the  produce  regulation  4)  of  diabetes,  Catecholamines that  the  decrease  alteration  interference  of  were  the  are  that  genetics  individual  segregated.  the  hepatic  species  selected  amongst not  observed  rats  are  used  worked  clearly  locus  We chose  with  in  the  binding DBA/2  experiments.  out  species  species  individual  the  the  other  mice,  in  defined,  which  these  the  HCLA  (C57BL/6 due t o  well  in  to  HCLA  rats, see  we if  binding  because  of  used there  the  known d i f f e r e n c e s  so-called that the mice  T.C.D.D  there  and  system, receptor protein  guinea while  in  all  association HCLA b i n d i n g binding  protein  the  since  have  a high  The  three  lack  species the  protein.  It  for that  receptor  indicates  specific  receptor  The  receptor  low  T.C.D.D  the  HCLA  there  binding  is  not  s y s t e m and  the  that  protein.  the  and  C57BL/6  T.C.D.D.  a very  indicates  also  may be a r a t  affinity  the  suggestion  this  1983).  evidence  T.C.D.D.  controls  was a  between  posess  of  which  there  (Finlayson,  DBA/2 mice  between  protein  Ah l o c u s  relationship  pigs  system.  the  receptor,  may be an  HCLA b i n d i n g  in  HCLA  an  6  PROPOSED  As  p r e v i o u s l y  hormonal  mentioned,  treatments  t e s t o s t e r o n e )  s i n c e  weeks  HCLA  f o r  the  in  amount  of  time  l e v e l s  of  the  the  undertaken i t  can  w i t h i n  be  to  (with  conscious  t a i l  d e l i n e a t e  i t s  see  r e g u l a t i o n  see  how  q u i c k l y  o p e r a t i o n i f  there  examine p r o t e i  what  the  in  be  e f f e c t  the  the  be  of  and  be  c o n t r o l  on  on  the  a l t e r a t i o n s , s t r e s s  on  the  course  of be  and  i f  o c c u r r i n g  attempted to  be  and  the  in f u r t h e r  r e g u l a t i o n .  and  may  more  HCLA  before  span.  d i a b e t i c  having  s t r e s s e d  and  longer  occurs  c o n t r o l  a n i m a l ,  c o n t r o l  should  p r o t e i n  c a t e c h o l a m i n e s  performed  time  l e v e l  animals  b i n d i n g  in  the a  a l t e r a t i o n s  should  performed  takes  d e p r e s s i o n  time  HCLA  appear  t h a t  A  w i t h  two  hormones)  p r o t e i n  same  of  takes  to  be  r e g u l a t o r y  other  n o n - s t r e s s e d  f u r t h e r role  the  i t  c o u l d  damaged  the  any  i n j e c t e d  that  p r o t e i n  It  b i n d i n g  course  ( s p e c i f i c a l l y  r e g e n e r a t e d .  replacement  should are  with  before)  should  in  be  HCLA  role  Adrenalectomy  is  the  vein  shown  proper  can  time  undertaken  b i n d i n g  of  s l i g h t y  longer  r a t s .  the  c o r r e l a t e d  (or  to  been  has  d i a b e t i c s  T e s t o s t e r o n e  rats  i t  p r o t e i n  d e p r e s s i o n  a  be  female  male  EXPERIMENTS  should  estrogen  hyp o p h y s e c t o m i z e d mechanism  FUTURE  in  the  same animals  to  c o m p l e t e l y  estrogen  b i n d i n g  n.  Ligand d i a b e t i c  s p e c i f i c i t y  animals  a l t e r a t i o n s  in  to  the  see HCLA  s t u d i e s i f  0  there  b i n d i n g  should are  be  any  p r o t e i n  performed  on  the  fundamental system  other  than  see  reduced  capacity.  studied  using  their are  the  the  disease  have  to  chemical  same a s s a y  state  differences  would  Spontaneous  in  be used  or  of  the  examined  control  binding  protein  diffuse  in  possible  the  exact  protein  would  out.  is  exclusive  be of the  isolation,  sequencing  of  the  they  to  seen  Other  to  if  of  to  of  if  the  if  it  is  to  HCLA  the  estrogen  protein  in  should  of  the  HCLA more  implications  the  of  there  a result  see  experiments  of  these  be  characterization,  HCLA b i n d i n g  are,  if  should  or  have  light  localization  STZ o r  are  be  effects  differences  liver  distribution  should  the  they  tissues  would  in  if  there  animals the  rats  with  If  see  Enucleation  further  see  a r e due t o  which  interest  B.B.  to  states.  and d i a b e t i c  function.  over  two  diseases.  intracellular  developing Finally,  if  distribution,  its  those  investigated  pathologies in  system  parallel these  diabetic  as  to  examine  binding  controversy receptors. and be  amino  carried  acid  SUMMARY  STZ-induced of  the  diabetes  HCLA e s t r o g e n  without  affecting  insulin,  ineffective  were  administered  normal  the  in in  estrogen  binding  a direct  interplay  these  in  the  alterations lead  us t o  between  in  the  in  the  the  and  male to  that  there  that  it  of  the  do  binding  protein  patterns  the  several  other  is  not  HCLA  effect  of  estrogen the diabetes,  connection the  not  species  specific.  HCLA  there  in  and  was  they  duplicated  observed  protein  rat  HCLA  restore  binding  may be  of  liver  hormone  when  and t h e  no d i r e c t  rat  including  that  seen  capacity  growth  which  The l a c k  is  male  levels  hormones  they  in  restore  metabolism  metabolism  cytosol  to  restore  while  drug/steroid  drug  reduced  rat.  the  and  indicates  these  in  treatments  patterns  levels  between  HCLA e s t r o g e n  liver  indicating  protein  levels,  the  inability  HCLA e s t r o g e n  sex-dependent Also,  This  conclude  found  triiodothyronine,  treatments  protein  Hormonal  doses  diabetic  hormonal  binding  Kd.  a decrease  protein  restoring  physiology.  levels  binding  testosterone,  were  causes  in  the  rat.  detectable  examined  BIBLIOGRAPHY  Anderson,  D.C.  Clin.  Endocri n o l .  Appel , M . C . , R o s s i n i , Diabetologia 15:  3:  69 ,  A.A., Williams, 3 2 7 , 1978  1974  R.M.,  Like,  A.A.  A r i s o n , R.N., C i a c c i o , E . 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Steroid  Biochem.  16:  437,  1982  APPENDIX  1)  Saturation  The  data  saturation plots  in  indicate  curves  contained  curves  the  which  Results  that  specific, range  plot  generated  Figure  be  in  this  were  used  section  used  section.  concentration could  I  The  saturable was with  to  were  samples  generate  the  saturation binding  taking  Scatchard  curves  over  place  of  the  before  had  to  ligand a  Scatchard  confidence.  Al  3 Specifically  bound 3  concentration citrate  control  described (3————d)  ([  in  [  H]-estradiol) animal.  the  Methods  non-specific  experiment  and  H]-estradiol in  v_s 1 i g a n d tail  Incubations section.  bound.  representative  carried  (o  Graph of  vein  the  o)  shown  is  results  injected out  as  Specific of  a  bound,  single  found.  7  lO-i  0  5 0  I O O  FREE LIGAND  1 5 0  Cnmol/1)  2 0 0  1  Figure A 2  Specifically  bound  concentration diabetic in  the  and  ([ H ] - e s t r a d i o l )  animal.  Methods  non-specific  [ H ] - e s t r ad i ol  Incubations  section.  bound.  representative  of  (o  in  v_s  ligand  4 day  STZ-induced  were c a r r i e d o) S p e c i f i c  Graph  shown  the  results  is  of  out  bound,  a single  found.  as  described fci  •)  experiment  

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