Primary cortisol resistance associated with a
thermolabile glucocorticoid receptor in a patient
with fatigue as the only symptom.
M Brönnegård, … , S Werner, J A Gustafsson
J Clin Invest.
1986;
78(5)
:1270-1278.
https://doi.org/10.1172/JCI112711
.
We have studied a woman with an apparent receptor-mediated resistance to cortisol on the
basis of elevated 24-h mean plasma cortisol levels and increased urinary free cortisol.
Plasma ACTH concentrations were normal but she was resistant to adrenal suppression by
dexamethasone. No stigmata of Cushing's syndrome were seen. To study the proposed
end-organ resistance to cortisol, we examined the glucocorticoid receptor (GR) in
lymphocytes and in fibroblasts from this patient and from her son. Several molecular
properties of the GR of lymphocytes from the patient were indistinguishable from that of
normal control subjects. In thermolability assays, however, the patient's GR as well as her
son's GR showed a striking heat sensitivity at 40 degrees and 45 degrees C when
compared with GR from normal persons. In addition, data from the thermolability assays
correlated well with the lack at 45 degrees C of dexamethasone-induced decrease in in vitro
[3H]thymidine incorporation into lymphocytes derived from both patients.
Research Article
Find the latest version:
Primary Cortisol Resistance Associated with
a
Thermolabile Glucocorticoid
Receptor in
a
Patient with
Fatigue
as
the Only Symptom
M.
BronnqgArd,**
S.Wemer,§ and J.-A.Gustafsson*Departments of*Pediatrics and fMedical Nutrition, Karolinska Institute, Huddinge University Hospital, S-141 86 Huddinge; §Department ofEndocrinology, Karolinska Hospital, S-104 01 Stockholm, Sweden
Abstract
We have
studied
a womanwith
an apparentreceptor-mediated
resistance tocortisol
on thebasis
of elevated 24-h mean plasma cortisol levels andincreased urinary
freecortisol. PlasmaACTH
concentrations were normal but she was
resistant
to adrenalsuppression by dexamethasone.
Nostigmata of Cushing's
syn-drome were seen. To study theproposed end-organ resistance
tocortisol,
weexamined
theglucocorticoid
receptor(GR)
in lymphocytes and infibroblasts
from this patient and from her son.Several molecular
properties of the GR
oflymphocytes
from
the
patient
wereindistinguishable from
thatof
normal controlsubjects.
Inthermolability
assays,however,
thepatient's GR
as well as herson's GR
showed astriking heatsensitivity
at400
and
450C
when comparedwith GR from
normal persons. Inaddition,
datafrom
thethermolability
assayscorrelated
wellwith
thelack at450C
of dexamethasone-induced decrease
ininvitroIHlthymidine incorporation
intolymphocytes
derived from bothpatients.
Introduction
Glucocorticoid resistance in humans, characterized by
hyper-cortisolism without the
features
of
Cushing's
syndrome,
wasfirst
described in 1976
by
Vingerhoeds
et al.(1) in
a50-yr-old
manwith advanced hypertension and hypokalemic alkalosis.
Sub-sequentbiochemical (2)
andgenetical
(3) studies indicated that
the
patient
suffered from
a rarefamilial syndrome where
theprimary cortisol resistance
wasdue
to aglucocorticoid
receptor(GR)'
present atthe
normalconcentration but with decreased
affinity
for cortisol.
In
this report,
wedescribe
awomanwith primary
glucocor-ticoid resistance in whom the end-organ insensitivity
to cortisolapparently is
notdue to a decreasedconcentration
or ligandaffinity
of the
receptor.However, the
GRof the
woman as well asofher
son,who
did
notshow
sopronounced
clinical
symptoms asthose
of his mother, displayed
anincreased thermolability,
aphenomenon also
seenwith androgen
receptorsfrom
patientswith the testicular feminization syndrome
(4).Case
report
The
patient
was46 yr old when she consulted her local hospitalfor
symptomsof edema,
red, swollen hands, and a pronouncedfatigue. She had
noprevious
history
ofdisease.
ThemenopauseReceivedfor
publication
4April198Sand in revisedform 24 June 1986.1.Abbreviations used in this
paper:
GR,glucocorticoidreceptor, TA, triamcinolone acetonide.had
occurred 3 yrearlier.
The patient's mother had also had anearly
menopause. Thepatient's
seven younger sisters werehealthy.
Herfather had died from
acerebral hemorrhage at the ageof 61
yr. Thepatient's
mothersuffered
from diabetes,hy-pertension,
and
cardiac insufficiency.
The patient's only child, ason29
yrold, had
hadperiods of inexplicable
fatigue that had madehim
stay homefrom
school andwork.The
patient
was acleaning assistant
who had workedhalf-time
for the
previous 10
yrowing
to atendency
tofatigue,
inthis
case asymptomincluding
anincreased
needof
sleep and afeeling of chilliness
andof
feebleness. During the
courseof
in-vestigation
thecutaneous changes and edemavanished
when arecently
introduced cleaning
detergent wasavoided.
Thephysical
examination revealed nothing pathological.
Herweight
was 48 kg overthe
pastseveral
yearsand her
height 166
cm. Herblood pressure was100-105/60-65
mmHg,with
noorthostatism.
There
were noindications of anorexia
or mentaldepression.
Thefindings of
hercomplete blood count, electrolytes, eryth-rocytesedimentation
rate, serum Fe, cholesterol,triglycerides,
albumin, endogenous creatinine clearance, liver function
tests,thyroid-stimulating
hormone,
triiodothyronine, thyroxine,
pro-lactin,
luteinizing hormone, follicle-stimulating hormone,
and testosteronewereall normal
for her
age.Because
of the
patient's
slim
features,
Addison's disease
wassuspected
butthree
urinary
24-hcortisol values
werefound
tobe
two- tofour-fold increased.
Thepatient
wasreferred
tothe
DepartmentofEndocrinology, Karolinska Hospital, Stockholm
todetermine
whethershe had
Cushing's
syndrome.
Plain
x-raysof
theskull
andpituitary fossa, cisternography,
and
2 yrlater,
computed tomography scanning
of the
pituitary
region
yielded normal results
asdid sonography
andcomputed
tomography
scanning
of the
adrenals,
pancreas,and
liver.
Skel-etal
x-raysshowed
nosigns
of
osteoporosis.
Since the detection of
hypercortisolism,
the
patient
has been
observed for
4 yrduring
which her
clinical features
(no
stigmata
of
Cushing's syndrome
and
continuing
complaints of
chilliness
and of
fatigue)
have notchanged.
She has
continued
towork
half-time
asacleaner.
The 24-hcortisol excretions
havecon-stantly
been
two- tofourfold increased
during
the
periods
when
she
hasfelt
completely healthy
aswellasduring
periods
of
fatigue.
Three 24-hurine
samples from
herson werecollectedfor
thedetermination of cortisol.
Thepatient
hasnotreceived
atrial
of
glucocorticoid
treatmentin orderto seewhetherher symptoms wouldimprove.
Methods
Inthis studyhealthyblood donorswereusedascontrols.Ineach control
experiment, 100mlof
peripheral
bloodwasused from which thelym-phocyteswereisolated.
Isolation
of
human
lymphocytes
Freshbloodwasdiluted withabalanced salt solution
(14.3
mMTris,
98AM MgCl2,0.54mMKCI,
5AMCaCl2, 126mMNaCl,
and 5.6nMJ.Clin. Invest.
© TheAmericanSociety for ClinicalInvestigation,Inc.
0021-9738/86/11/1270/09
$1.00
D-glucose,pH7.4)andlayeredon adiscontinuous
Ficoll-Paque
gradient(Pharmacia,Uppsala, Sweden)insiliconized 50-mlcentrifugetubes. After centrifugation at 400 g in a Beckman TJ6 centrifuge (Beckman Instru-ments, Inc., Palo Alto, CA) for 30 min at 18'C,the layer containing
lymphocytes was withdrawn. The lymphocytes were washed twice by resuspension in the balanced salt solution andcentrifugationat 100 g for 10min at 18'C. More than 95% of thelymphocyteswereviable as
determinedbythe trypan blueexclusiontestandcountingina hemo-cytometer(Barker,KeboLaboratory, Stockholm) (5, 6).Cells thatwere notusedimmediatelywerefrozen and stored at-70'C.
Preparation
ofcytosol
Inorder toeliminatetheinfluenceofendogenous steroids in the Scatchard
analysiswe used the procedure described by Rosner and Polimeni(7).
They showed that during 20 h at00C,soluble enzymes in the cytosol almostcompletely metabolize cortisoltoproducts which donotinterfere with the receptor binding of
[6,7-3H]triamcinolone
acetonide (3H-TA: specificactivity300kBq/mmol,
Amersham SearleCorp., Amsterdam,theNetherlands).Atthe end of thistime,specificallybound 3H-TA (see below) is a valid measure ofthe total receptor concentration. Lymphocytes isolatedfrom fresh blood were kept inamedium (RPMI 1640)without serum,supplemented withL-glutamine (2 mM), bensyl-penicillin (400
IU/ml), andstreptomycin(0.2mg/ml),for 24 hat0C. Thelymphocyte suspensionwasthencentrifugedand thepelletwasstoredat-70°Cuntil usedfor thepreparationof cytosol.
Fibroblastsfromestablished cell cultures (described below)growing
in monolayers were kept in a medium(minimalessentialmedium) with-out serum, supplementedwithL-glutamine (2 mM),bensyl-penicillin
(400 IU/ml), andstreptomycin(0.2 mg/ml), for 24 h at37°C.The me-dium was then removed, the monolayers wererinsed, the cells were harvestedwithtrypsin-EDTA,and theresulting cell suspension was
cen-trifuged.The cell pelletwasstoredat-70°C until used for thepreparation
of cytosol.
Humanlymphocytesandfibroblasts storedat-70°Cwerelysedby
thawingat4°C. Cells were then homogenized inice-cold ETGbuffer
(1 mM EDTA, 20 mMTris-HCI,pH7.8, 10% (wt/vol)glycerol,and 2 mMdithiothreitol)inanall-glassDounce Bhomogenizer
using
20 manual strokes. Thehomogenateswerecentrifugedat 100,000g for 30 minat0°CinaBeckman type 50 SWrotor(Beckman Instruments, Inc.,Palo Alto,CA). The clear supernatantswereused as cytosol. The cytosol was
incubatedwith 100 nM
3H-TA
at0°Cfor 4 h in all experiments except in the case of the Scatchardanalysis,where theincubation period was 24 h.A200-fold molar excess of unlabeledTAwas used in order to determine specific(i.e., displaceable) binding.Whole cell binding
Thebinding capacityof mononuclear leucocytes was determined by
in-cubatinglymphocyteswith medium without serum for 24 h and then washing the cells twice with phosphate-buffered saline before adding
var-iousconcentrationsof3H-TA in 2 ml of medium without serum and with or without a 200-fold molar excess of nonradioactive TA. Each
incubationcontained 106cells/ml medium and was performed at 37°C ina5%CO2incubator for 90 min. The medium was removed by
cen-trifugation,the cellswerewashedtwice withphosphate-bufferedsaline, and harvested on glass-fiber filter-papers for measurement of
radio-activity. Binding capacity, expressed as number of binding sites per
cell, and apparent dissociation constant were calculated according to Scatchard (8).
Cell culture
Fibroblastswereestablished from explants of skin. Punch biopsies, 4
mmdiam, were taken from the patient and her son and from six con-trols. Fibroblast strains and cell cultures were established according to Ham (9).
Monolayer
binding
Cells from stock flasksweredissociated with 0.05%
trypsin-EDTA
atroomtemperature for5min and seeded(day0)at aconcentration of
50,000
cells per3 ml ofmediumcontaining
10%(vol/vol)
fetal calfserum,
supplemented
withL-glutamine (2 mM), bensyl-penicilhin
(400
IU/ml),andstreptomycin
(0.2
mg/ml),in each well ofa24-wellplastic
plate
(Chemoferm Inc., Stockholm).
Onday
7themonolayerswererinsed with 3 ml ofphosphate-buffered saline,and 3mloffresh medium withoutserum wasadded.Onday8the mediumwasremoved,andthe
mono-layerswererinsed twice with 3mlof medium withoutserumandthen incubated with various concentrations of 3H-TA in medium without
serumand withorwithouta200-foldmolarexcessof nonradioactive TA.Thisprocedureallowsmeasurementof maximum
glucocorticoid-binding capacity
of the cells in the absence ofendogenoussteroids. In-cubationswereperformed
at370C
ina5%CO2
incubator for 90min. Themediumwasthenremoved,
themonolayers
wererinsed,
and the cellswereharvestedwithtrypsin-EDTA,
andfinally
collectedonglass-fiberfilter-paperswithacellharvester formeasurementofradioactivity. Binding capacity, expressed as number of binding sites per cell,
and apparent dissociation constant were calculated according to
Scatchard
(8).
Scatchard
analysis
ForScatchardanalysis(8)the cytosol prepared from lymphocytes and from fibroblastswasincubated withincreasingconcentrations(3.1-50 nM)of 3H-TA alone orinthe presence ofa200-fold molarexcessof unlabeled TA.Atthe end of the incubationperiod,20-24 hat0-40C, aliquots of 10
gl
weretaken for determination of total radioactivity.Bound andfree steroidwasseparatedbytheaddition ofonethird of the incubation volume ofa suspension of dextran-coated charcoal (final
concentration1%(wt/vol)charcoal and 0.1%(wt/vol) dextran) (10).The
mixturewasleft for 10 minat0C and then centrifugedat400 g for 10 min.Specific bindingwasdefinedasthedifference in boundradioactivity
between samples containing labeled steroid only(total binding) and samples containing both labeled andexcessunlabeled steroid(nonspecific binding).
Isoelectric
focusing
in
polyacrylamide gel
Asafurthermeans toquantitatethe GRweusedisoelectric focusing in polyacrylamide gel according to Hanssonetal. (6).Polyacrylamide gels for isoelectric focusingwerepreparedasdescribed by Wrange (I 1). All samples were appliedinacrylic glass frames and analyzed simultaneously. The isoelectricfocusingwas performedat a currentof 20 mA anda
voltageof 1,200 V. The total running time was 90 min. The gel strip
correspondingtothesamplewas cutinto 0.5-cm slices and transferred
tocounting vials. After addition of 5 ml of scintillator fluidtothevials,
the radioactivity in the sliceswasextractedat60°C for 45min,and the radioactivitywasmeasured inaliquid scintillation counter. Hanssonet
al.(6)alsodemonstratedabetter recovery of total steroid-receptor
com-plexes if the isoelectric focusingwascarried out after limitedproteolysis
of the receptor. In the present experiments trypsin (81 U/mg of protein,
i.e.,0.8
Ug/(A2M-A310)
unit,seebelow)wasused(10) and the reactionwascarriedoutfor 30 minat10°C.
Protein
determination
Anestimation of the cytosolic protein concentration was obtained by expressing the difference in optical densityat280nmand310 nm, (A2m-A310).Proteinwasalso determined according to Lowry et al. (12). Receptor concentrationswereexpressedasfemtomoles per milligram of protein.
DNA-cellulose chromatography
DNA-cellulose waspreparedasdescribed by Alberts and Herrick (13). Cytosolic samples (1 ml) were labeled for 4 h at 0°C using 100 nM final concentration of 3H-TA. Labeled cytosolwastreated with dextran-coated charcoal to remove unbound steroid (10) and activation of the steroid-receptor complex was performed by incubation at 25°C for 30 min. The incubation mixture was cooled on ice and applied on a 1-ml
DNA-cellulose column. The column wasincubated with the sample for 15
min and then washed with five to six column volumes of ETG buffer. The columnwaselutedwith5 mlof ETG buffer containing 0.3MNaCG. Aliquots, 100 Ml were taken from each fraction and countedfor radio-activity.
Gelfiltration
Gelfiltration wascarried out on Agarose A-1.5 (Bio-Rad Pharmacia, Uppsala). Samplesof 3 ml were applied on the column (87 X 1.5 cm) and chromatographed at 10 cm/h in ETG buffer; 0.15 MKCl;0.02% (wt/vol) NaN3; 20 mM molybdate. Molybdate was added to the sample 30 minafter the addition of3H-TAand 1 hbefore application to the column. The column wascalibrated using the following proteins: horse spleenferritin,
14C-methylated
bovine serum albumin, '4C-methylatedcatalase, and'4C-methylated whale skeletal muscle myoglobin. The Stokes radii of these proteins are 6.15, 3.59, 5.20, and 2.01 nm (14, 15), re-spectively.
Hormone assays
Serumcortisol (16),urinaryfree cortisol (17), ACTH (18), aldosterone (19), insulin (20), and C peptide (21)weredetermined by radioimmu-noassays.Cortisol-binding globulin and sex hormone-binding globulin weredetermined byequilibrium dialysis (22). The urinary excretion of glucocorticoid precursors and metaboliteswasdetermined by high-pres-sureliquid chromatography (23).
Assays
for
temperature
stability of
GR
Whole cell assay. Fibroblasts were grown in monolayer cultures as de-scribed above. Onedaypriortotheexperimentthemonolayerswere
rinsed with 2 ml ofphosphate-buffered saline and 3 ml of fresh medium (minimal essentialmedium) withoutserumwere added.Inexperiments
assessing bindingat
400
and450C,
themonolayerswerepreincubatedat400or45°Cfor 60 min in medium withoutserum.Themediumwas
then removed, and medium containing 100nM 3H-TA± a200-fold molarexcessof unlabeledTA(warmedtoindicatedtemperatures)was
added. Themonolayers were then incubated for 60 min in theCO2
incubatorat400or45°C,respectively.Inexperiments assessing binding
at300 and35°C, medium with 3H-TA± a200-fold molarexcessof unlabeledTA(warmedto300or350C)wasaddedtothe monolayers, and themonolayerswereincubated inaCO2incubatorat300 or350
for 60 min. Afterincubationattheindicated temperatures, the medium wasremoved, themonolayerswererinsedtwice,and thecellswere har-vestedwithtrypsin-EDTA, then centrifugedandhomogenized before
aliquotsweretakenformeasurementof
radioactivity
andprotein. Bindingwasexpressedasnumberofspecific bindingsites permilligram protein.
There was nodetectable metabolism of3H-TAafterwarmingof the incubation mixtureto
450C
asassessedbythin-layer chromatography.Cytosolicassay. Afterhomogenizationandcentrifugationofa
sus-pensionof
lymphocytes
orfibroblasts,the supernatant(i.e.,
cytosol)wasremoved and used forbindingassay.Bindingwasperformedat
40C
for 30 min,using 100 nM3H-TA+a200-fold molarexcessof unlabeled TA.Priortoincubationofcytosolathighertemperatures thesamplesweretreated with dextran-coated charcoal in orderto removefreesteroid,
asdescribed in the Scatchard
analysis
above. Theincubationswerethen continued for 60 minat250, 300,
350, 400,
and450C,
respectively.
After 90 min the incubationswereterminated and thecytosol
treated with one-third volume of dextran-coated charcoal(cf. above).
Specific binding
wasdetermined andexpressedasfemtomoles of3H-TAbound per mg ofcellularprotein.
Thymidine
incorporation
Invitrosensitivity of cellstoglucocorticoidswasmeasuredbystudying
theeffects of dexamethasoneonincorporationofradiolabeledthymidine.
Lymphocytes (5X 106/ml)wereincubated intriplicatewithorwithout
101
M dexamethasone for 24h at370C.
Radiolabeled thymidine([methyl-3H]thymidine,
specific activity2.9 TBq/mmol, AmershamIn-ternational,Amersham,Buckinghamshire, England; final concentration 2 kBq
[methyl-3H]thymidine/ml)
was thenadded and the incubationwascontinued for 6 h atindicated temperatures. The cells were harvested
onglass-fiberfilter-paper,andisotope incorporationwasdetermined by liquid scintillation counting. Results from in vitro sensitivity studieswere
expressedaspercentchange of cellularisotopeincorporationaftersteroid
treatment.
Results
Glucocorticoid
tests.Diurnal
serumcortisol, which
wasanalyzed onthreeoccasions, showed slightly elevated levels andabluntedrhythm (Fig. 1 a). InFebruary 1982, determination of diurnal
plasma ACTHconcomitantly withserumcortisolgavethe values
6, 4, 4, 2, 3, and 20 pmol/liter, respectively; these figuresare
normal when compared with the reference ACTH valueat6
a.m.,30±26pmol/liter. Sporadic plasma ACTH analyzed
con-comitantly with serum cortisol at 8 a.m. on four occasions
showed ACTHlevels ranging between 8 and 38 pmol/liter with
serumcortisolranging between550and780nmol/liter (reference value80-550nmol/liter). 1nmol cortisol/literwasequalto3.62
,ug
cortisol/I00
ml(the molecular weight for cortisol is 362,500).Cortisol-binding globulinwas691nmol/liter, corresponding
touppernormalrangelevels foryoungwomen(22). Sex
hor-mone-binding globulinwas102nmol/liter(reference value
67-137nmol/liter).
The combined dexamethasone suppression (1 mgorally)/
ACTH stimulation (25 IU i.v.)test showeddecreased cortisol suppressionand normal cortisolincrements(Fig. 1 b).
The low- andhigh-dose oral dexamethasone suppressiontest,
illustrated in Table I, showed decreased cortisol inhibition to
exogenousglucocorticoids (24).
Themetyraponeadministration (750mgX6/24 h)test
pro-vokedafourfold increment of basal Porter-Silber chromogens
from 8, 7, and 8 mg/d (reference value 3-10 mg/d)to33 mg/d
on theday of metyraponeadministration, 25 mg/d onday 1,
and 13 mg/don day 2 after metyrapone administration. The
responseindicated intact corticotropin-releasing hormone and
ACTH reactivity and slightly hyperreactive adrenal glands (24). Oral
glucose loading.
The oralglucose
load showed normal fasting glucose levels and a normal postprandial pattern, the lowest glucoselevelbeing 2.4 mmol/literat210min.Concom-itantanalysisofplasmainsulin and Cpeptideshowednormal
fasting
levels and theexpectedrises afteroralglucose challenge;Cpeptideand insulin levelswere0.4pmol/ml(referencevalue 0.18-0.63pmol/ml)and 8
,U/ml
(referencevalue 19.6±9.8,U/
ml), respectively, under basalconditions,2.6pmol/ml and72
gU/ml
at30min,
and 3.2pmol/mland 126,gU/ml
at60min. Hence, therewere nosignseither ofdecreasedsensitivitytoin-sulin as seenin truehypercortisolism orincreasedsensitivityto
insulin,
whichshouldhavebeenexpectedina caseofclinicallyovertcortisol resistance.
Mineralocorticoid axis. Diurnal
plasma aldosterone
wasnormal. Urinaryaldosterone, serum and urinary sodium and
potassium,andplasmarenin inrecumbent andupright positions
werenormal,as were serum bicarbonateand chlorides. There
was on nooccasionanytendencytohypokalemiaoralkalosis.
Theseobservationsarenotinaccordance with thecasepreviously
describedby Vingerhoeds etal.(1) where thepatient suffered
fromhypertensionaswellasfromseverehypokalemia.
Excretion
ofurinary
steroids.Analysis
wasmade when total24-h cortisolwas765nmol/d (referencevalue83-300 nmol/d).
The steroid excretoryprofilewasnormal; the 24-h excretion of
androsterone was 3.7 ,mol, etiocholanolone 5.9 umol, dehy-droepiandrosterone2.1,mol, 1-oxo-etiocholanolone 2.7 Mmol,
lB-hydroxyandrosterone
+ 17a-hydroxypregnanolone
3.9,umol,
I1gB-hydroxyetiocholanolone
4.6Amol,
pregnanediol2.1,umol,
andpregnanetriol0.8gmol.
Theexcretion of the cortisol metabolites tetrahydrocortisone, tetrahydrocortisol,8.00 1200 1M00
0
CL
w
z
0 >j
N
4C w
x
Jz
X- W
E In
4c
z
V- N
... ...I...
22.00 2.00 6.00 8.00 HOURS
8.00 23.00 8.00 9.00 9 30 1000
day day 2
Figure 1. (a) Diurnal serum corti-sol inawomanwith increased uri-nary excretion of cortisol without clinicalsignsofhypercortisolism.
Cortisol levelswereanalyzedin (o) February and (c) June 1982 and in (A) February 1984. Shadedarea
de-notes serumcortisol(mean±2 SD;
n=28) in healthy controls.
(b)
Serum cortisol before and 9 h after
bv
1 mg of dexamethasone and 1.5b
and 2 h after an intravenous injec-tion of 25IUofsyntheticACTH (1-24 Synacthen). Shadedareade-11.00
12.00
HOURS notes normalresponse(mean±2 SD;n=32).(i)
Patient.Abbrevia-> tion: 1, liter.
werewithin the normal totalrange,20.2
gmol/d.
However,theratios betweendifferent cortisol metabolitesweresimilartothose
seeninhypercortisolismduetoCushing'sdisease(22); tetrahy-drocortisol/tetrahydrocortisonewas 1.2(reference< 1) and
tet-rahydrocortisol/allotetrahydrocortisolwas4.7(reference<4.5).
Studiesonthepatient'sson.One ofthreeurinary24-hcortisol
valueswasincreased,381 nmol/d (referencevalue formen 83-300nmol/d).Theresults of thecompletebloodcount,
electro-lytes,liver andrenal function tests, serumcholesterol, and
tri-glycerideswerewithin normallimits. Hewasasymptomaticwith
nostigmataofCushing's syndrome.
Scatchardanalysis.Thespecific bindingof3H-TAincytosol
from thepatient's lymphocytesaswellasfromcontrol
lympho-cytesis shown inFig. 2,where the inset shows data replotted accordingtoScatchard(8).Thestraightlines obtained(r=-0.80
andr=-0.82)suggestthat,underourassayconditions, 3H-TA Cortisol Resistance AssociatedwithGlucocorticoidReceptor 1273
N
0
0
th
k-09
0-To
900
800
,00
-600
-
500-
400-
300-
200-100
-
0-1100
-000
-900
-800
-700 -1-%
E
-wi
0
W
600
-500
-400
-300
-200
-100
Table I. Serum and Urinary Cortisol during Peroral Suppression Test,withLow andHigh Doses ofDexamethasone
Day 1 2 3 6 7 10 11
Dexamethasone
0.5mgX4, day 4-7 + +Dezamethasone
2 mgX4, day 8-11 + +Serum cortisol,
nmol/liter
725 365 442 40 30Urinary
cortisol,
nmol/liter 604 765 590 132 153 36 48binds
to asingle class of receptor sites of
uniform ligandaffinity,
both in
lymphocytes from
thepatient
and inlymphocytes from
thecontrols.
The totalnumber
of binding sites per cell was 1,600and
5,400 (patient:
twodeterminations)
and3,500±1,300
(con-trols:
n=8). The dissociation constants
were 4.2 and 3.9(patient:
twodeterminations) and
4.1±2.0 nM(controls:
n=8). The son showedsimilar
GRcharacteristics
as assayed inlymphocytes.
The total numberof binding
sites per cell was 2,000 and 1,500(two determinations) and
thedissociation
constant was 4.5 and5.3
nM(twodeterminations).
Inaddition,
the bindingcapacity
of
culturedskin fibroblasts, expressed either
asfemtomoles
permilligram of
protein in cytosol or sites per cell(RO)
in wholecells
did
notreveal
anydifferences between
the twopatients
andthe controls.
Inall
cases,the
total number of binding sites
percell
wasapproximately
40,000 and
theapparent dissociation
constant5.0 nM. The results from
ourstudies
on thecharac-teristics
of
the GRfrom
thepatient,
herson, andcontrols
aresummarized
inTable
II.From these results
it
isobvious
that nodifferences
existcon-perning
receptor-ligand affinity
or numberof specific
ligand-binding
sites in
lytnphocytes
orfibroblasts
whencomparing
the twopatients with
thecontrols.
According
toLippman
and Barr(25),
nodifferences
wereseenbetween
subpopulations
of
humanmononuclear
cells with
regard
-to KD values ornumber ofspecific glucocorticoid binding
sites
percell.
Therefore,
the unfractionated lymphocytes isolated
with
Ficoll-Paque should contain
ahomogeneous pool of
GR.
DNA-cellulose chromatography.
The DNA-binding
capacity
of
lymphocytic
GR
wasstudied
using
DNA-cellulose
chroma-tography of thermally activated
receptor(26).
Inorder
to eluteX 100
LU
<800.
Z= 600.
CD60
560
10004I
0 BOUNDTA(pM/ml
Lj 'Co
LLJ
,.3
200I/
00>
50 100FREE TA ( nM )
Figure 2. Binding of 3H-TA to cytoplasmic receptors from(o)the pa-tient and from (x) controls. Cytosol was incubated with various con-centrations of 3H-TA as described in Methods. (Inset) Scatchard plot of the binding data.
(@)
Patient (mean of three determinations of each concentration); shaded area denotes the range of the eight controls.both the 6- and 3.3-nm
receptorforms,
previously described for
the
ratliver
GR andshown
toelute
at0.17 and 0.25
MNaCl,
respectively (27), DNA-cellulose
wasfirst washed
withfive
tosix column volumes of ETG buffer, giving the flow-through
con-taining
nonactivated GR, and then eluted with
5ml ETG buffer
with 0.3 MNaCl.
Nosignificant difference
intotalDNA-cellulose
binding of activated
lymphocytic
GR from
thepatient
ascom-pared
tothe controls
wasfound
(Table III).
Gelfiltration.
Inorder
tocomparefurther the GR from the
patient and controls, respectively, agarose filtration
ofmolybdate-stabilized
cytosolic
ligand-receptor complex
wascarried
out.GR from human lymphocytes,
particularly
inits free
form,
isgenerally
unstable
incytosol, probably due
to ahigh
concentra-tion of
proteolytic
enzymesin
this
typeof
preparation
(28).
Inorder
tostabilize
the receptorduring long
chromatographic
pro-cedures,gel filtration
wascarried
outin the
presenceof
molyb-date.Table
IVsummarizes
theresults from the gel filtrations.
The8.
1-nm
nonactivated complex
aswell
as the 5.1-and the
3.6-nmcomplex
wererepresented
bothin cytosol from
thecontrols
and in
the
cytosol from
thepatients.
These
results areingeneral
accordance with findings
previously
reported by
Sherman et al. (28,29)
onmolybdate-stabilized GR
in
cytosolic preparations
from
avariety of tissues.
In
conclusion,
nodifference
insize
between GRcomplexes
from
thepatient and the controls,
respectively,
wasfound.
Temperature stability.
Inthecytosolic
assay very
lowlevels
of
specific 3H-TA binding,
2.2±1.9
and
0.3±0.5
fmol/mg
protein,
respectively,
werefound
at400
and
450C
in
lymphocytes
from
the
patient.
Incontrols,
onthe other
hand,
the 95% confidence
interval for
specific binding
at40'C
was17.8-66.9
fmol/mg
protein and
at45°C 16.1-22.7
fmol/mg protein (Fig.
3
c).
A
dramatic
drop
inspecific
3H-TA
binding
in
cytosolic samples
from
thepatient
occurred
between
350
and
40°C. Incubations
of
cytosol from
thepatient
and thecontrols
respectively,
at0°C
up to35°C
did
notreveal
anydifferences
inspecific binding
of
3H-TA.
Similar
profiles
of GR
thermolability
curves wereobserved
for fibroblasts.
Incontrols
the95% confidence interval
for
specific
cytosolic
binding
at40°C
was210-285fmol/mg protein
andat45°C 95-135
fmol/mg
protein (Fig.
3
a).
The
patient
had anabnormally
low3H-TA-binding capacity
at400
and45°C,
7.5±3.5
fmol/mg protein
andundetectable binding,
respectively.
Even at
30° and 35°C,
thepatient
showed lowerspecific
3H-TAbinding
than the controls. Hersonalso showedanabnor-mally
low3H-TA-binding capacity
atthesetemperatures,
125+7
fmol/mg
protein
at40°C
andundetectable
binding
at45°C.
At30°C, however,
he
still showedthe
same3H-TA-binding
capacity
asthe
controls.
Comparable results
wereobtained
with the whole cell assay but inthis
casethespecific
binding
of
thesoncompared
to that of thecontrols
didnotdiffer until the temperature
wasTable II. Concentration andLigand
Affinity
ofGR in Patients and ControlsMother* Son* Controls (mean±SD)
Wholecells
Cultured skin fibroblasts
Bindingcapacity
(R.),
sites percell 35,000; 40,000 25,000;35,000
40,000±15,000
(n=6)Apparentdissociationconstant,nM 5.0;3.5 4.5;8.0 7.0±2.5(n=6)
Mononuclearleukocytes
Binding capacity
(R.),
sitespercell 1,600;5,400 2,000; 1,500 3,300±1,300(n=8)Apparentdissociationconstant,nM 8.0;5.3 4.0;4.5 5.5±2.0(n= 8)
Cytosol
Culturedskin fibroblasts
Binding capacity
(R.),
98; 100 80;65 120±30(r=-0.89±0.05) (n=6)fmol/mg protein (r =-0.90; -0.89) (r =-0.93; -0.90)
Dissociationconstant, nM 9.5;8.3
9.8;
7.4 7.5±2.5(n
=6)Mononuclearleukocytes
Bindingcapacity
(R.),
110;390 190;280 430±120(r=-0.89±0.03)
(n=8)fmol/mg protein (r =-0.80;-0.82) (r=-0.82; -0.90)
Dissociationconstant,nM 4.2;3.9 5.3;4.5 4.1±2.0
(n
=8)Preparationofcytosolandincubation of cellswerecarriedout asdescribed in Methods. Abbreviation:r,correlation coefficient of the Scatchard regression lines. *Results fromtwoseparatedeterminations.
interval for
specific
whole cellbinding
at40°C
was65-85fmol/
mgprotein
and at45°C
10-20fmol/mg protein.
Thepatient
had undetectablebinding
at40°C
aswell as at45°C,
whereas hersonhada3H-TA-bindingcapacity
of 5±3fmol/mg protein
at40°C
andundetectablebinding
at45°C. Incubations ofcytosol
from fibroblasts and whole fibroblasts at 0°C up to 30°C did notreveal anydifferences
inspecific binding of
3H-TAbetween thetwopatients
and thecontrols, respectively (data
notshown).
Inmixed cytosolic
samplesfrom
acontrol andthe twopa-tients specific
binding
wasdetectable
even at45°C,indicating
that
thedisappearance of
binding
in samplesfrom
thepatients
at
elevated
temperatures wasnotdue totheactivation of
pro-teases(Fig.
4aandb).
Furthermore,addition
of proteaseinhib-itors
tocytosolic
incubations did
not alter thethermolabilityprofiles of specific
3H-TAbinding for
any of the two patients(data
notshown).
Wealsoinvestigated
theeffect of lowering the temperature used in themonolayerbinding assay to 25°C aftera
preincubation
at45°Cfor
60 min. In these experiments bothpatients
showedareversible
decrease in specific binding at ele-vated temperature (data not shown). Taken together, these resultsindicate
the presence of a thermolabile GR in cells from theTable III. DNA-cellulose Chromatography of Thermally Activated Human
Lymphocytic
Glucocorticoid ReceptorFlow-through Retained
Patient(n = 2) 86;82 13; 15
Control (n =6)* 75±1.4 19±1.2
Theflow-throughrepresentsthefraction ofthe complex detectedafter washing theDNA-cellulosecolumn with 5-6 vol of ETG buffer
with-outsalt. Recoveryin percentageof totally appliedcomplex. The
re-tained
3H-TA
receptorcomplexwaseluted with 5 mlof0.3 M NaCl in ETGbuffer.Forfurtherdetails,see text.*Allvalues represent
mean±SD.
patient
aswellasfrom herson.The defectisprobably
intrinsic inthe receptormolecule
andis not related to the cellular milieu.Thymidine incorporation. The inhibition of [3H]thymidine
incorporation
inlymphocytes
at350,
370,
and45°C
was morepronounced in controls thaninthetwopatients (Table V).At
45°C,
noinhibitionwasobserved in eitherpatient;
these resultsare inagreement with the loss ofspecific 3H-TAbindingatthese
temperatures incells from the patients
(cf.
above).Discussion
The
patient
understudy
showedanapparentend-organ
insen-sitivity
tocortisol.Despite signs
of chronicendogenous
hyper-cortisolism-increased serum andurinary cortisol,
alteredex-cretory
profiles
of cortisolmetabolites, hyperreactive
adrenalre-sponse aftermetyrapone
administration,
and decreasedcortisolTableIV.Stokes
Radius
(Rs)*ofMolybdate-stabilized Glucocorticoid Receptorfrom
thePatientand Controls,asDetermined
by
Gel FiltrationonAgaroseA-1.5.PatientRs(n=2) ControlsRs(n=5)t
nm nm
8.1;8.3 8.1±0.2
5.1; 5.3 5.1±0.3
3.6;3.6 3.7±0.2
Cytosolswereprepared from lymphocytes,asdescribedinMethods,
andgel filtration ofthe
3H-TA-labeled
cytosolswascarriedout on AgaroseA-1.5.Samples of3 ml wereappliedonthe columnandchro-matographedat10 cm/h in ETG buffer; 0.15M
KCl;
0.02% (wt/vol) NaN3; 20mMmolybdate.*Stokesradius (Rs)asdetermined byagarosefiltrationandlinear regression analysis of logRsvs.distribution coefficient fortheinternal standards.
tAllvaluesrepresentmean±SD.
;i4
3-c
3
.2
IV1
._
0-c4
.0
CO-EE
2
Q
30' 45*
Temp.(C)
Figure4.Analysisofproteolyticactivity. Cytosolwaspreparedas de-scribedinMethods.0.5mlof3H-TA-labeledcytosoIfrom(n)a
con-trolwasmixedatindicatedtemperatureswith0.5 ml of unlabeled
cy-tosol from (A)the motherorwith0.5mlof unlabeled cytosol from (o)
theson.Afterincubationfor 60min,themixturesweretreated with dextran-coatedcharcoal andcentrifuged and aliqotsweretaken for
measurementofradioactivity. Using thesamecytosolicpreparations,
specificbindingat300and450Cwasdetermined by incubating1 ml
ofcytosolfrom eachpatient,as well asfrom the control, with 100 nM 3H-TA±a200-fold molarexcessof nonradioactive TA. The values
representthemeanoftWo separatedeterminations and the specific 3H-TA-binding capacityaftermixingthecytosolisindicatedwitha
solidline(C). (a) Mixing experiment (mother). (b) Mixing
experi-ment(son). Temp.(C)
Figure 3. Effect of elevatedtemperatureonspecificbinding of 3H-TA
inlymphocytes and fibroblasts from (A) the patient, (o) herson,and
(*)controls. At each temperaturespecific 3H-TA-binding capacitywas
determinedontriplicate aliquotsofcytosolorwells ofmonolayers,as
describedinMethods, and themean±SDwascalculated fromthese threeincubations. Thisprocedurewasrepeatedfor each of the 10
con-trols andatotal mean±SDwascalculated for each temperature. For thepatientand hersonspecific 3H-TA-binding capacitywa's
deter-minedin thesame mannerandrepeatedtwice.(a)Cytosolic
assay-fibroblasts.(b)Whole cellassay-fibroblasts. (c) Cytosolic assay-lym-phocytes.
inhibition byexogenousglucocorticoids-shelacked the classical features ofCushing's syndrome.Incontrast, she hadsignsand symptoms reminiscent of Addison'sdisease, namely,lowbody weight,low bloodpressure,andfatigue.Therewas noclear
ev-idenceof advanced adrenalhyperplasia: sonographyand
com-puted tomography scanningvisualized adrenalglandsof normal
size.DiurnalplasmaACTHwaswithin the normalrange.
Thus,inthis patienttheperipheral insensitivitytocortisol
was notassociated with the secondary, ACTH-dependent in-creased secretion of mineralocorticoids foundinthepreviously reportedcase(2)withveryadvanced cortisol resistanceand with
cortisol levels similartothose ofourpatient.This indicates the existence ofsubgroupsofprimaryeortisolresistance withvarying
compensatory mechanisms. Because of the relatively small numberoffamilymembersinvestigated (cf. Fig. 5)nopattern
TableV.Inhibitionof
[HI
Thymidine Incorporation at Indicated TemperaturesTemperature +dex -dex Percent change
dpm/mgprotein dpm/mgprotein
150C
Controls 280±20 620±20 55±10(n=6)
Mother* 225;330 450;660 50;50
Son* 290;255
605;
510 52;50350C
Controls 180±15 600±30 70±15(n=6)
Mother* 340;315 610;450 45;30
Son* 290;300 485;545
40;
45370C
Controls 210±25 555±30 65±10(n=6)
Mother* 405;390 605;590 33; 34
Son* 350;390 620;605 43;36
450C
Controls 280±30 430±30 35±10(n=6)
Mother* 550;485 555;480 5;ND
Son* 395;430
405;
455 2;5Incubation of cells and determination of
[3H]thymidine
incorporationwerecarriedout asdescribed in Methods. Abbreviations:dex, dexa-methasone;
ND,
nodetectableinhibition.* Results fromtwoseparatedeterminations.
1276 M.Bronnegdrd,S. Werner, and J.-A.Gustafsson
- 4
rC=17 4'-a
I
_ Xn
3
.- 1
U1
ncx > 2
E
'i.
._k_
a
a. -
&.s
-*osvs
~~~~'
30*
45-b
" \0
I"ss
2
61 62 64 65
6667
111 ~~~~11
Figure 5. Family pedigree.The father(I; 1)of thepatient (II; 3)died
attheageof 61 inacerebralhemorrhage.Themother of thepatient (I; 2)issufferingfromhypertension,cardiacinsufficiency,and diabetes
mellitus. Three brothers andthree sisters of thepatientareallhealthy
withnoknown endocrinologicdiseases. Theson(III; 1)of thepatient
hasmarkedlyelevatedurinaryfree cortisol concentrations and shows thesametendencyoffatigueashismother.
of inheritancemaybepredictedatthisstage. Inthepresentcase, amoderate increase in cortisol secretionwassufficientto main-tainanadequate peripheral action of cortisol, asindicatedby normal glucose, insulin, and C-peptide increments afteranoral glucose load.
Thepresenceof normal concentrations ofcytosolic GR with
normal ligand affinity in the patient's lymphocytes and fibroblasts
wasevidentlynotsufficient foranormalresponsivenessto glu-cocorticoids. Thereareseveralsteps after the initialbindingof
thesteroidtothereceptorthatprecede the phenotypic expression of the steroid action characteristic ofagiven celltype. Any of these biochemicalevents,including the activation ofthe
steroid-receptorcomplex, its subsequent translocation tothe nucleus, and
specific
bindingtoDNA,maybe the site ofadefect, possiblybecause of structural changes in thereceptormolecule. Interestingly, thesonof the patient also showedaGR
ther-molability butanotherwise normalreceptor status.Therewas,
however,aminor difference in thermolability between mother
andson,the latter showingaslightly less thermolabilereceptor attemperaturesranging from 30° to40°C. Clinically, this bio-chemical observation issupported by the fact that thesondoes
haveatendency offatigue butnot tosuchanextentashis mother.
Furthermore,lymphocytes from the controlswereglucocorticoid
responsive at 45°C, as indicated by the suppressive action of
dexamethasoneonincorporation of [3H]thymidine into the cells,
whereasnodexamethasone-induced decrease in
[3H]thymidine
incorporation in vitrowas observed in lymphocytesfrom the
twopatientsatthistemperature;these results correlate well with thedata from the GRthermolabilityassays.
Primary cortisol resistanceorspontaneoushypercortisolism withoutCushing's syndromewasfirst described by Vingerhoeds
etal. in 1976 (1). These authors postulatedahyposensitivityto
glucocorticoid action inahypertensive, hypokalemic, but
oth-erwisehealthy male patient. The patient hada veryhigh cortisol
production rate with elevated plasma ACTH. Further studies
onthe cortisol resistance of this patientwerepublished in 1982
by Chrousos et al. (2, 3), this time with regard to the ligand affinity of GRaswellastothe GRnumberin whiteblood cells
andfibroblasts. Incontrast toourfindings, theseauthors reported
adecreased ligand affinity andareducedreceptornumber,
con-cluding that the cortisol resistancewasduetoanabnormal GR
with decreased cortisol affinity.
Receptor-related glucocorticoid resistance because of
"ac-tivation-labile"receptorcomplexes has beenreportedinhuman
mutant lymphoma cell lines(30). Furthermore, therapy with
pharmacologic glucocorticoid doses without the development
of the stigmata of
Cushing's
syndrome has been linked with steroid resistance in receptor defective lymphocytes in patients with acute leukemia (31). Principally, it appears that alterationsin
thesensitivity
toglucocorticoids
can be traced tobiochemical
lesions at the receptor level (4, 32). In some cases, however,unresponsiveness
toglucocorticoids
occursdespite seemingly
normal receptorfunctions and receptorconcentration (33).
Mostof these observations,
our ownfindings,
and some other reports onsteroid action in humans (33, 34) imply that the
clinicalmanifestations of steroid resistance in
general
arerarely,
if ever, due tothe
absence of a steroid hormonal receptor, butareprob-ably due
tochanges
in the receptor molecule that prevent its normalfunction.
The
finding of
atemperatureinstability
of GR in lympho-cytes and fibroblasts fromourpatients
suggests the presence of aminor structural
abnormality
inthe
receptor molecule. Such aminor
protein abnormality has been
described for enzymes inbacterial
mutants in which an elevation oftemperature isas-sociated with altered
growth (35).
Furthermore,
in mutatedmammalian
cellsstructurally abnormal
enzymes have beenfound
toberesponsible
for the effects of elevatedtemperature
onthe
functions ofthe cells
(36, 37).
An amino acid substitution atanyposition
inthe
protein might
leadto analteration in thetertiary
structureof the
protein
and
consequently
toalteredbio-chemical
properties.
Griffin (4) has described
athermolabile
androgen
receptorin
cultured humanfibroblasts from
patients
with testicularfem-inization.
According
tohim,
thedegree of feminization
waspos-itively
correlated to thebiochemical alterations
atthe receptorlevel.
In our case, thesteady-state clinical
picture
was that ofintermittent
subtleglucocorticoid insufficiency-low
weight,
mild
hypotension, and intermittent
fatigue.
Acknowledaments
Wewish to thank Leslie Morgan for her secretarial assistance. This studywassupportedbyagrant from the Swedish Medical Re-searchCouncil (No. 13x-2819).
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