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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:

(2)

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 woman

with

an apparent

receptor-mediated

resistance to

cortisol

on the

basis

of elevated 24-h mean plasma cortisol levels and

increased urinary

freecortisol. Plasma

ACTH

concentrations were normal but she was

resistant

to adrenal

suppression by dexamethasone.

No

stigmata of Cushing's

syn-drome 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 astriking heat

sensitivity

at

400

and

450C

when compared

with GR from

normal persons. In

addition,

data

from

the

thermolability

assays

correlated

well

with

thelack at

450C

of dexamethasone-induced decrease

ininvitro

IHlthymidine incorporation

into

lymphocytes

derived from both

patients.

Introduction

Glucocorticoid resistance in humans, characterized by

hyper-cortisolism without the

features

of

Cushing's

syndrome,

was

first

described in 1976

by

Vingerhoeds

et al.

(1) in

a

50-yr-old

man

with advanced hypertension and hypokalemic alkalosis.

Sub-sequent

biochemical (2)

and

genetical

(3) studies indicated that

the

patient

suffered from

a rare

familial syndrome where

the

primary cortisol resistance

was

due

to a

glucocorticoid

receptor

(GR)'

present at

the

normal

concentration but with decreased

affinity

for cortisol.

In

this report,

we

describe

awoman

with primary

glucocor-ticoid resistance in whom the end-organ insensitivity

to cortisol

apparently is

notdue to a decreased

concentration

or ligand

affinity

of the

receptor.

However, the

GR

of the

woman as well as

ofher

son,

who

did

not

show

so

pronounced

clinical

symptoms as

those

of his mother, displayed

an

increased thermolability,

a

phenomenon also

seen

with androgen

receptors

from

patients

with the testicular feminization syndrome

(4).

Case

report

The

patient

was46 yr old when she consulted her local hospital

for

symptoms

of edema,

red, swollen hands, and a pronounced

fatigue. She had

no

previous

history

of

disease.

Themenopause

Receivedfor

publication

4April198Sand in revisedform 24 June 1986.

1.Abbreviations used in this

paper:

GR,glucocorticoidreceptor, TA, triamcinolone acetonide.

had

occurred 3 yr

earlier.

The patient's mother had also had an

early

menopause. The

patient's

seven younger sisters were

healthy.

Her

father had died from

acerebral hemorrhage at the age

of 61

yr. The

patient's

mother

suffered

from diabetes,

hy-pertension,

and

cardiac insufficiency.

The patient's only child, ason

29

yr

old, had

had

periods of inexplicable

fatigue that had made

him

stay home

from

school andwork.

The

patient

was a

cleaning assistant

who had worked

half-time

for the

previous 10

yr

owing

to a

tendency

to

fatigue,

in

this

case asymptom

including

an

increased

need

of

sleep and a

feeling of chilliness

and

of

feebleness. During the

course

of

in-vestigation

thecutaneous changes and edema

vanished

when a

recently

introduced cleaning

detergent was

avoided.

The

physical

examination revealed nothing pathological.

Her

weight

was 48 kg over

the

past

several

years

and her

height 166

cm. Herblood pressure was

100-105/60-65

mmHg,

with

no

orthostatism.

There

were no

indications of anorexia

or mental

depression.

The

findings of

hercomplete blood count, electrolytes, eryth-rocyte

sedimentation

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 testosteronewere

all normal

for her

age.

Because

of the

patient's

slim

features,

Addison's disease

was

suspected

but

three

urinary

24-h

cortisol values

were

found

to

be

two- to

four-fold increased.

The

patient

was

referred

to

the

Department

ofEndocrinology, Karolinska Hospital, Stockholm

to

determine

whether

she had

Cushing's

syndrome.

Plain

x-rays

of

the

skull

and

pituitary fossa, cisternography,

and

2 yr

later,

computed tomography scanning

of the

pituitary

region

yielded normal results

as

did sonography

and

computed

tomography

scanning

of the

adrenals,

pancreas,

and

liver.

Skel-etal

x-rays

showed

no

signs

of

osteoporosis.

Since the detection of

hypercortisolism,

the

patient

has been

observed for

4 yr

during

which her

clinical features

(no

stigmata

of

Cushing's syndrome

and

continuing

complaints of

chilliness

and of

fatigue)

have not

changed.

She has

continued

to

work

half-time

asa

cleaner.

The 24-h

cortisol excretions

have

con-stantly

been

two- to

fourfold increased

during

the

periods

when

she

has

felt

completely healthy

aswellas

during

periods

of

fatigue.

Three 24-h

urine

samples from

herson werecollected

for

the

determination of cortisol.

The

patient

hasnot

received

a

trial

of

glucocorticoid

treatmentin orderto seewhetherher symptoms would

improve.

Methods

Inthis studyhealthyblood donorswereusedascontrols.Ineach control

experiment, 100mlof

peripheral

bloodwasused from which the

lym-phocyteswereisolated.

Isolation

of

human

lymphocytes

Freshbloodwasdiluted withabalanced salt solution

(14.3

mM

Tris,

98AM MgCl2,0.54mM

KCI,

5AMCaCl2, 126mM

NaCl,

and 5.6nM

J.Clin. Invest.

© TheAmericanSociety for ClinicalInvestigation,Inc.

0021-9738/86/11/1270/09

$1.00

(3)

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: specificactivity300

kBq/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 minat

0°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

at

roomtemperature for5min and seeded(day0)at aconcentration of

50,000

cells per3 ml ofmedium

containing

10%

(vol/vol)

fetal calf

serum,

supplemented

with

L-glutamine (2 mM), bensyl-penicilhin

(400

IU/ml),andstreptomycin

(0.2

mg/ml),in each well ofa24-well

plastic

plate

(Chemoferm Inc., Stockholm).

On

day

7themonolayerswererinsed with 3 ml ofphosphate-buffered saline,and 3mloffresh medium without

serum 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-cubationswere

performed

at

370C

ina5%

CO2

incubator for 90min. Themediumwasthen

removed,

the

monolayers

were

rinsed,

and the cellswereharvestedwith

trypsin-EDTA,

and

finally

collectedon

glass-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 reaction

wascarriedoutfor 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.

(4)

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-methylated

catalase, 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

and

450C,

themonolayerswerepreincubated

at400or45°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. Binding

wasexpressedasnumberofspecific 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)was

removed and used forbindingassay.Bindingwasperformedat

40C

for 30 min,using 100 nM3H-TA+a200-fold molarexcessof unlabeled TA.Priortoincubationofcytosolathighertemperatures thesamples

weretreated with dextran-coated charcoal in orderto removefreesteroid,

asdescribed in the Scatchard

analysis

above. Theincubationswerethen continued for 60 minat

250, 300,

350, 400,

and

450C,

respectively.

After 90 min the incubationswereterminated and the

cytosol

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 at

370C.

Radiolabeled thymidine

([methyl-3H]thymidine,

specific activity2.9 TBq/mmol, Amersham

In-ternational,Amersham,Buckinghamshire, England; final concentration 2 kBq

[methyl-3H]thymidine/ml)

was thenadded and the incubation

wascontinued 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

serum

cortisol, which

wasanalyzed onthreeoccasions, showed slightly elevated levels andablunted

rhythm (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 oral

glucose

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

at30

min,

and 3.2pmol/mland 126

,gU/ml

at60min. Hence, therewere nosignseither ofdecreasedsensitivityto

in-sulin as seenin truehypercortisolism orincreasedsensitivityto

insulin,

whichshouldhavebeenexpectedina caseofclinically

overtcortisol resistance.

Mineralocorticoid axis. Diurnal

plasma aldosterone

was

normal. 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 total

24-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

+ 1

7a-hydroxypregnanolone

3.9

,umol,

I1gB-hydroxyetiocholanolone

4.6

Amol,

pregnanediol2.1

,umol,

andpregnanetriol0.8

gmol.

Theexcretion of the cortisol metabolites tetrahydrocortisone, tetrahydrocortisol,

(5)

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 1.5

b

and 2 h after an intravenous injec-tion of 25IUofsyntheticACTH (1-24 Synacthen). Shadedarea

de-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,the

ratios 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

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-100

(6)

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 30

Urinary

cortisol,

nmol/liter 604 765 590 132 153 36 48

binds

to a

single class of receptor sites of

uniform ligand

affinity,

both in

lymphocytes from

the

patient

and in

lymphocytes from

the

controls.

The total

number

of binding sites per cell was 1,600

and

5,400 (patient:

two

determinations)

and

3,500±1,300

(con-trols:

n=

8). The dissociation constants

were 4.2 and 3.9

(patient:

two

determinations) and

4.1±2.0 nM

(controls:

n=8). The son showed

similar

GR

characteristics

as assayed in

lymphocytes.

The total number

of binding

sites per cell was 2,000 and 1,500

(two determinations) and

the

dissociation

constant was 4.5 and

5.3

nM(two

determinations).

In

addition,

the binding

capacity

of

cultured

skin fibroblasts, expressed either

as

femtomoles

per

milligram of

protein in cytosol or sites per cell

(RO)

in whole

cells

did

not

reveal

any

differences between

the two

patients

and

the controls.

In

all

cases,

the

total number of binding sites

per

cell

was

approximately

40,000 and

the

apparent dissociation

constant

5.0 nM. The results from

our

studies

on the

charac-teristics

of

the GR

from

the

patient,

herson, and

controls

are

summarized

in

Table

II.

From these results

it

is

obvious

that no

differences

exist

con-perning

receptor-ligand affinity

or number

of specific

ligand-binding

sites in

lytnphocytes

or

fibroblasts

when

comparing

the two

patients with

the

controls.

According

to

Lippman

and Barr

(25),

no

differences

were

seenbetween

subpopulations

of

human

mononuclear

cells with

regard

-to KD values or

number ofspecific glucocorticoid binding

sites

per

cell.

Therefore,

the unfractionated lymphocytes isolated

with

Ficoll-Paque should contain

a

homogeneous pool of

GR.

DNA-cellulose chromatography.

The DNA-binding

capacity

of

lymphocytic

GR

was

studied

using

DNA-cellulose

chroma-tography of thermally activated

receptor

(26).

In

order

to elute

X 100

LU

<800.

Z

= 600.

CD60

560

1000

4I

0 BOUND

TA(pM/ml

Lj 'Co

LLJ

,.3

200I/

00>

50 100

FREE 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

receptor

forms,

previously described for

the

rat

liver

GR and

shown

to

elute

at

0.17 and 0.25

M

NaCl,

respectively (27), DNA-cellulose

was

first washed

with

five

to

six column volumes of ETG buffer, giving the flow-through

con-taining

nonactivated GR, and then eluted with

5

ml ETG buffer

with 0.3 M

NaCl.

No

significant difference

intotal

DNA-cellulose

binding of activated

lymphocytic

GR from

the

patient

as

com-pared

to

the controls

was

found

(Table III).

Gelfiltration.

In

order

tocompare

further the GR from the

patient and controls, respectively, agarose filtration

ofmolybdate-stabilized

cytosolic

ligand-receptor complex

was

carried

out.

GR from human lymphocytes,

particularly

in

its free

form,

is

generally

unstable

in

cytosol, probably due

to a

high

concentra-tion of

proteolytic

enzymes

in

this

type

of

preparation

(28).

In

order

to

stabilize

the receptor

during long

chromatographic

pro-cedures,

gel filtration

was

carried

out

in the

presence

of

molyb-date.

Table

IV

summarizes

the

results from the gel filtrations.

The

8.

1-nm

nonactivated complex

as

well

as the 5.1-

and the

3.6-nm

complex

were

represented

both

in cytosol from

the

controls

and in

the

cytosol from

the

patients.

These

results arein

general

accordance with findings

previously

reported by

Sherman et al. (28,

29)

on

molybdate-stabilized GR

in

cytosolic preparations

from

a

variety of tissues.

In

conclusion,

no

difference

in

size

between GR

complexes

from

the

patient and the controls,

respectively,

was

found.

Temperature stability.

Inthe

cytosolic

assay very

low

levels

of

specific 3H-TA binding,

2.2±1.9

and

0.3±0.5

fmol/mg

protein,

respectively,

were

found

at

400

and

450C

in

lymphocytes

from

the

patient.

In

controls,

on

the other

hand,

the 95% confidence

interval for

specific binding

at

40'C

was

17.8-66.9

fmol/mg

protein and

at

45°C 16.1-22.7

fmol/mg protein (Fig.

3

c).

A

dramatic

drop

in

specific

3H-TA

binding

in

cytosolic samples

from

the

patient

occurred

between

350

and

40°C. Incubations

of

cytosol from

the

patient

and the

controls

respectively,

at

0°C

up to

35°C

did

not

reveal

any

differences

in

specific binding

of

3H-TA.

Similar

profiles

of GR

thermolability

curves were

observed

for fibroblasts.

In

controls

the

95% confidence interval

for

specific

cytosolic

binding

at

40°C

was210-285

fmol/mg protein

andat

45°C 95-135

fmol/mg

protein (Fig.

3

a).

The

patient

had an

abnormally

low

3H-TA-binding capacity

at

400

and

45°C,

7.5±3.5

fmol/mg protein

and

undetectable binding,

respectively.

Even at

30° and 35°C,

the

patient

showed lower

specific

3H-TA

binding

than the controls. Hersonalso showedan

abnor-mally

low

3H-TA-binding capacity

atthese

temperatures,

125+7

fmol/mg

protein

at

40°C

and

undetectable

binding

at

45°C.

At

30°C, however,

he

still showed

the

same

3H-TA-binding

capacity

asthe

controls.

Comparable results

were

obtained

with the whole cell assay but in

this

casethe

specific

binding

of

theson

compared

to that of the

controls

didnot

differ until the temperature

was

(7)

Table II. Concentration andLigand

Affinity

ofGR in Patients and Controls

Mother* 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 cell

binding

at

40°C

was65-85

fmol/

mg

protein

and at

45°C

10-20

fmol/mg protein.

The

patient

had undetectable

binding

at

40°C

aswell as at

45°C,

whereas hersonhada3H-TA-binding

capacity

of 5±3

fmol/mg protein

at

40°C

andundetectable

binding

at

45°C. Incubations ofcytosol

from fibroblasts and whole fibroblasts at 0°C up to 30°C did notreveal any

differences

in

specific binding of

3H-TAbetween thetwo

patients

and the

controls, respectively (data

not

shown).

In

mixed cytosolic

samples

from

acontrol andthe two

pa-tients specific

binding

was

detectable

even at45°C,

indicating

that

the

disappearance of

binding

in samples

from

the

patients

at

elevated

temperatures wasnotdue tothe

activation of

pro-teases

(Fig.

4aand

b).

Furthermore,

addition

of protease

inhib-itors

to

cytosolic

incubations did

not alter thethermolability

profiles of specific

3H-TA

binding for

any of the two patients

(data

not

shown).

Wealso

investigated

theeffect of lowering the temperature used in themonolayerbinding assay to 25°C after

a

preincubation

at45°C

for

60 min. In these experiments both

patients

showeda

reversible

decrease in specific binding at ele-vated temperature (data not shown). Taken together, these results

indicate

the presence of a thermolabile GR in cells from the

Table III. DNA-cellulose Chromatography of Thermally Activated Human

Lymphocytic

Glucocorticoid Receptor

Flow-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 defectis

probably

intrinsic inthe receptor

molecule

andis not related to the cellular milieu.

Thymidine incorporation. The inhibition of [3H]thymidine

incorporation

in

lymphocytes

at

350,

370,

and

45°C

was more

pronounced in controls thaninthetwopatients (Table V).At

45°C,

noinhibitionwasobserved in either

patient;

these results

are inagreement with the loss ofspecific 3H-TAbindingatthese

temperatures incells from the patients

(cf.

above).

Discussion

The

patient

under

study

showedanapparent

end-organ

insen-sitivity

tocortisol.

Despite signs

of chronic

endogenous

hyper-cortisolism-increased serum and

urinary cortisol,

altered

ex-cretory

profiles

of cortisol

metabolites, hyperreactive

adrenal

re-sponse aftermetyrapone

administration,

and decreasedcortisol

TableIV.Stokes

Radius

(Rs)*ofMolybdate-stabilized Glucocorticoid Receptor

from

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 columnand

chro-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.

(8)

;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 Temperatures

Temperature +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;50

350C

Controls 180±15 600±30 70±15(n=6)

Mother* 340;315 610;450 45;30

Son* 290;300 485;545

40;

45

370C

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;5

Incubation of cells and determination of

[3H]thymidine

incorporation

werecarriedout 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

n

cx > 2

E

'i.

._

k_

a

a. -

&.s

-*osvs

~~~~'

30*

45-b

" \0

I"ss

(9)

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, possibly

because 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 alterations

in

the

sensitivity

to

glucocorticoids

can be traced to

biochemical

lesions at the receptor level (4, 32). In some cases, however,

unresponsiveness

to

glucocorticoids

occurs

despite seemingly

normal receptorfunctions and receptor

concentration (33).

Most

of these observations,

our own

findings,

and some other reports on

steroid action in humans (33, 34) imply that the

clinical

manifestations of steroid resistance in

general

are

rarely,

if ever, due to

the

absence of a steroid hormonal receptor, butare

prob-ably due

to

changes

in the receptor molecule that prevent its normal

function.

The

finding of

atemperature

instability

of GR in

lympho-cytes and fibroblasts fromour

patients

suggests the presence of a

minor structural

abnormality

in

the

receptor molecule. Such a

minor

protein abnormality has been

described for enzymes in

bacterial

mutants in which an elevation oftemperature is

as-sociated with altered

growth (35).

Furthermore,

in mutated

mammalian

cells

structurally abnormal

enzymes have been

found

tobe

responsible

for the effects of elevated

temperature

on

the

functions ofthe cells

(36, 37).

An amino acid substitution atany

position

in

the

protein might

leadto analteration in the

tertiary

structure

of the

protein

and

consequently

toaltered

bio-chemical

properties.

Griffin (4) has described

a

thermolabile

androgen

receptor

in

cultured human

fibroblasts from

patients

with testicular

fem-inization.

According

to

him,

the

degree of feminization

was

pos-itively

correlated to the

biochemical alterations

atthe receptor

level.

In our case, the

steady-state clinical

picture

was that of

intermittent

subtle

glucocorticoid 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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