Increased proteolysis An effect of increases in plasma cortisol within the physiologic range

Increased proteolysis. An effect of increases in

plasma cortisol within the physiologic range.

P S Simmons, … , J E Gerich, M W Haymond

J Clin Invest.

1984;

73(2)

:412-420.

https://doi.org/10.1172/JCI111227

.

Prolonged exposure to glucocorticoids in pharmacologic amounts results in muscle

wasting, but whether changes in plasma cortisol within the physiologic range affect amino

acid and protein metabolism in man has not been determined. To determine whether a

physiologic increase in plasma cortisol increases proteolysis and the de novo synthesis of

alanine, seven normal subjects were studied on two occasions during an 8-h infusion of

either hydrocortisone sodium succinate (2 micrograms/kg X min) or saline. The rate of

appearance (Ra) of leucine and alanine were estimated using [2H3]leucine and

[2H3]alanine. In addition, the Ra of leucine nitrogen and the rate of transfer of leucine

nitrogen to alanine were estimated using [15N]leucine. Plasma cortisol increased (10 +/- 1

to 42 +/- 4 micrograms/dl) during cortisol infusion and decreased (14 +/- 2 to 10 +/- 2

micrograms/dl) during saline infusion. No change was observed in plasma insulin,

C-peptide, or glucagon during either saline or cortisol infusion. Plasma leucine concentration

increased more (P less than 0.05) during cortisol infusion (120 +/- 1 to 203 +/- 21 microM)

than saline (118 +/- 8 to 154 +/- 4 microM) as a result of a greater (P less than 0.01) increase

in its Ra during cortisol infusion (1.47 +/- 0.08 to 1.81 +/- 0.08 mumol/kg X min for cortisol vs.

1.50 +/- 0.08 to 1.57 +/- 0.09 […]

Research Article

Find the latest version:

http://jci.me/111227/pdf

Increased

Proteolysis

An Effectof Increases in Plasma Cortisol within the

_{Physiologic Range}

Patricia S. Simmons, JohnM. Miles, JohnE. Gench,

and MoreyW. Haymond

Endocrine Research Unit, Departments of Pediatrics, Medicine,

andPhysiology, Mayo Clinic andFoundation, Rochester, Minnesota55905

As

bstract. Prolonged

exposure

to

glucocorti-coids in

pharmacologic

amounts

results

in

muscle wasting,

but

whether changes in

plasma

cortisol within the

phys-iologic

range

affect

amino

acid

and

protein

metabolism

in man has not been

determined.

To

determine

whether

a

physiologic

increase in plasma cortisol increases

pro-teolysis

and the

de

novo

synthesis

of

alanine,

seven

normal

subjects

were

studied

on two

occasions during

an

8-h

infusion of

either hydrocortisone sodium succinate (2

_1g/

kg

.

min) or

saline.

The rate

of

appearance

(Ra) of

leucine

and

alanine

were

estimated using

[2H3]leucine

and

[2H3]alanine.

In

addition,

the Ra

of

leucine nitrogen

and

the

rate

of

transfer of leucine nitrogen

to

alanine

were

estimated using

['5N]leucine.

Plasma

cortisol increased (10±1 to 42±4

1Ag/dl)

during

cortisol infusion and decreased

(14±2

to

10±2

gg/dl)

during

saline

infusion.

No

change

was

observed in

plasma

insulin,

C-peptide,

or

glucagon during

either

saline or

cortisol

infusion. Plasma

leucine concentration increased

more

(P

<

0.05)

during cortisol infusion (120±1

to

203±21

uM)

than

saline (118±8

to

154±4

_jAM)

as a

result

of

a

greater

(P

<

0.01)

increase in its Ra

during

cortisol

infusion (1.47±0.08 to 1.81±0.08

gmol/kg

-

min

for

cor-tisol

vs.

1.50±0.08

to

1.57±0.09

,mol/kg

min).

Leucine

nitrogen

Ra

increased

(P

<

0.01) from

2.35±0.12

to

3.46±0.24

,umol/kg

min, but less

so

(P

<

0.05)

during

saline

infusion (2.43±0.17

to

2.84±0.15

Amol/kg

min,

Thiswork was presented, inpart, at the 63rd Annual Meeting of the

EndocrineSociety,Cincinnati, OH, June, 1981.

Address reprint requests to Dr. M. W. Haymond, Mayo Clinic,

EndocrineResearchUnit,Rochester, MN 55905.

Receivedfor publication 6 June 1983 and in revisedform 4 October

1983.

P

<

0.01). Alanine Ra increased (P

<

0.05) during cortisol

infusion but remained

constant

during

saline

infusion.

During

cortisol, but

not

during

saline

infusion,

the rate

and percentage of leucine nitrogen going

to alanine

in-creased (P

<

0.05).

Thus,

an

increase

in

plasma

cortisol within the

phys-iologic range increases proteolysis and

thede novo

syn-thesis of alanine,

a

potential

gluconeogenic substrate.

Therefore, physiologic changes in plasma cortisol play

a

role

in

the

regulation

of whole

body protein

and

amino

acid

metabolism

in man.

Introduction

Prolonged administration of pharmacologic amounts of

glu-cocorticoidsin man causes musclewasting (1).Bothstimulation ofproteolysis and inhibition ofprotein

synthesis

have been implicated in this lossofbody protein. The proteolytic effect ofpharmacologicamountsofglucocorticoidsonskeletalmuscle is evidenced by increases in (a) efflux of amino acids from incubatedratmuscle(2-4);(b)plasmaaminoacid concentrations

ineviscerated animal preparations(5,6);(c) plasmaureanitrogen (2); (d)muscledipeptidaseactivity(7); and (e)3-methylhistidine excretion (8).In contrast,nosignificant increaseinprotein deg-radation wasobserved in rat musclefollowingseveral days of highdose invivocorticosteroneexposure(9-11). Inhibition of protein synthesis also has been invoked to explain theprotein catabolic state induced by steroids on the basis ofdecreased

incorporation of amino acids into ratmuscle following gluco-corticoid treatment (9-12).

Inman, increases inurinarynitrogen excretion and plasma concentrations of alanine and glutamine are observed only after 24-48 h of exposure to pharmacologic doses of glucocortico-steroids(13, 14). These data suggest that several days may be required before evidence of a glucocorticoid effect on amino acid metabolism can be seen in man. Whether short-term changes inplasma cortisol concentration within the physiologic rangeaffect protein metabolism in man has not been determined. Infusion of cortisol to achieve high physiologic concentrations

increases the plasmaconcentrations of phenylalanine and the

J.Clin. Invest.

© The American Societyfor Clinical Investigation, Inc.

0021-9738/84/02/0412/09 $1.00

branched-chain amino acids, leucine, isoleucine,andvaline (15). An increase in the plasma concentration of these essential amino acids in the postabsorptive statecould result from eitherincreased proteolysis or decreased utilization (oxidation and/or protein synthesis). Since the branched-chain amino acids are an

im-portantnitrogen source for synthesis of the potential gluconeo-genic substrate alaninein vivo(16-18), cortisol stimulation of

proteolysis could increase alanine availability via denovo

syn-thesis, as well as directly from protein. The present studieswere

designed to determine if increases of plasma cortisol within the physiologic range could increase the rate ofappearance ofleucine, anindicator of whole body proteolysis, and,ifso, to determine the effect of increased availability of leucine on the de novo

synthesisof alanine.

Methods

Subjects.Informedconsentwasobtainedfromsevenhealthy adult

vol-unteers between the agesof 23 and 38 yr. All were within 10% of ideal body weight(Metropolitan Life Insurance Table) and none had a family history of diabetes mellitus. The subjects had consumed a weight-main-taining diet conweight-main-tainingatleast200 g of carbohydrateforaminimum of 3 d before study.Allvolunteers wereadmitted to the Clinical Study Unit on theafternoon before study and were provideda600-calmeal

at 1800 h. On the evening before study, an 18-gauge plastic catheter was inserted into a forearm vein for isotope infusion.

Experimental design. Each subject wasstudied ontwo occasions separated by at least2wk, once during infusion of hydrocortisonesodium

succinate (2pg/kg.min)beginning at 0 time, and on a second occasion during saline infusion. At -8 h,a constantinfusion ofL-['5NJleucine

was begun at a rate (0.14

_Amol/kg

*min) toenrich the circulating plasma pool of leucineto'-5mol%andwascontinuedtothe endof the study. Isotopic equilibrationof'5Ninto the alanine pool has been demonstrated

tooccurinthisperiod of time(18).On themorning of study,aprimed

constantinfusionofL-[2H3]leucine(0.04umol/kg*min),L-[2H3]alanine

(0.15

Imol/kg-

min), and

[3H]glucose

(3,800dpm/kg.min) was begun

at-4 h andcontinued throughout the study. At zerotime (I 100 h),

an infusion of either salineorcortisol (2 Mg/kg.min)wasbegun and continued throughout the studyperiod(hour 8).

Arterializedvenous blood wasobtained froma 19-gauge scalp vein needle placed retrograde in the contralateralhandvein (19, 20). Blood sampleswere obtained at -2, -1.5, -1, -0.5, 0, 0.5, and 1 h, and subsequently at 1-h intervals through hour 8.

Approximately 17 mlof bloodwere drawnateachsamplingand

aliquotedinthefollowingfashion:5 ml wereaddedto atubecontaining

EDTA andbenzamadine for assay of glucagon,insulin, C-peptide, and cortisol; 10 mlwere addedto atube containingsodium heparin for amino acid and substrate analysis; and2 ml wereadded to atube containing sodium fluoride for determination ofglucose and glucose specific activity.Alltubes wereplacedonice,centrifugedat4°C, and the plasma storedat-70°C until assay. Statistical analyseswerecarried

outusingapairedt test.

Analytical methods.

L-[15N]Leucine, L-[5,5,5-2H3]leucine,

and

L-[3,3,3-2H3]alanine(Merck, Sharp&Dohme, IsotopeDivision, Montreal, Canada)weredetermined tobepyrogen-freeand >99% pure. Sterile,

pyrogen-free D-[3-3H]- and

D-[2-3H]glucose

wereobtained fromNew

England Nuclear(Boston, MA).

Plasmaleucineconcentrationsweredetermined by gas

chromatog-raphy/mass spectrometry(GC/MS)' utilizing[2H7Jleucine as an internal standard (21).Infusate amino acid concentrations were determined by ion exchange chromatography (BeckmanI19 CL Autoanalyzer, Beckman Instruments, Inc., Fullerton, CA) (22). Plasma alanine,

D-f-hydroxy-butyrate, acetoacetate, lactate, and pyruvate were determined by mi-crofluorometric enzymatic techniques (23-25). Plasma insulin (26), glu-cagon (27), andC-peptide (28) were measured by radioimmunoassay. Plasma cortisol was determined by protein-binding assay (29) and plasma FFA were determined colorimetrically (30).

Plasma glucose was measured using a YSI glucose analyzer (Yellow Springs Instrument Co., Yellow Springs, OH) and[3H]glucose radio-activity was determined by the methodof Dunn et al. (31). The metabolic clearance rate for glucose was calculated by dividing the rate of dis-appearance by theplasmaconcentration. Isotopic enrichment in plasma leucine and alanine was determined by GC/MS as previously described (17, 32) using the trimethylsilyl derivatives and a 2 mX 2-mm 3% OV 11 (Supelco, Inc., Bellefonte, PA) packed columninterfaced by jet sep-arator to a5985B Hewlett Packard GC/MS (Hewlett-Packard Co., Palo Alto, CA). Moles percent enrichment of[I5N]alanine, [2H3]alanine,

['5N]leucine,

[2H3J1eucine,

and[2H7Jleucinewere calculated bycomparing the peakheight ratio oftheionsof interest fromtheplasmasamples with those obtained on the same day from a standard curve of known enrichment. Actualratesof stable isotopeinfusion were determined as the product of the infusate amino acid concentration(micromoles per milliliter), isotopeenrichment, and infusion rate (milliliter per minute). Apparentratesof glucose appearance (Ra) anddisappearance (Rd) were calculated from the equationsof Steele (33), modified for nonsteady-state conditions (34). Apparent rates of appearance (Ra) and disap-pearance (Rd)for alanine and leucine were determined using Steele's equationsfor nonsteady-state conditions adapted for stable isotopic tracers (18, 35). The assumptions and equations for estimating Ra, Rd, and nitrogen exchange used have been previously described(16-18). Apparent rates of leucine appearance and disappearance calculated from

[2H3J1eucine

wereusedtodetermine leucine carbon(C) kinetics, since

this tracer provides rates ofleucine flux similar to those obtained with

[U-'4C]leucine(21),

[1-'4C]leucine

(36), and

[1_'3C]leucine

(37).Rates

of appearance of[I5Njalanine, percentage of alanine nitrogen derived from leucine, and the percentage and rate of leucine nitrogen transfer

toalaninewerecalculatedusing standard product precursor relationships (16-18).

Results

Plasma hormoneconcentrations. Duringthe infusion of hydro-cortisone, plasmacortisol concentrations increased(P<0.001)

from 10±1 sg/dlto 42±4

_Ag/dI

by hour 3, and subsequently remained nearly constant. During infusion ofsaline, plasma cortisol concentrations decreasedfrom 14±2jsg/dlto9.5±2

_,g/

dl (P<0.05)athour8(Fig. 1).Plasma concentrations ofinsulin

(9.0±0.2 ,U/ml and 9.1±0.2

gU/ml),

C-peptide (1.7±0.1 and

1.9±0.1

Ag/ml),

andglucagon(287±5and298±12pg/ml)were notsignificantly differentbefore infusion of saline or cortisol

anddidnotchange subsequently (Fig. 1).

Effects ofcortisolon leucine metabolism. Plasma leucine 1.Abbreviations usedinthis paper:GC/MS,gaschromatography-mass

spectrometry; KIC,a-ketoisocaproate; Ra, rate(s) of appearance; Rd, rate(s) ofdisappearance.

N 7 |C

tlr

8ae; MEAN*SEM

40

20

oL

20

_4f 10

D

Plasma Cortisol

*p<o*cf base line

tp<0.01

saline vs.cortisol

Plasma Insulin

-?'

L

4 - Plasma

_C-peptide

a

o

L

500 Plasma Glucagon

Zig

300

&4a

100 , i , * .

-2 0 2 4 6 8

Hours

Figure1.Plasmacortisol, insulin, C-peptide,andglucagon

concentra-tion

during

saline

_(.)

orcortisol

(o)

infusion insevennormaladults.

concentration increased 70% (120±1to203±21 ,uM,P<_0.01)

during cortisol,butonly30%(118±8to 154±4

_IM,

P<0.05) during saline infusion. Althoughbase-line leucine valueswere notdifferentonthetwostudydays, plasmaleucine concentration

washigher(P<0.05) by hour3 ofthe cortisol when compared with thatof the saline infusion and remainedsofor theduration

ofstudy (Fig.2).

During cortisol infusion, therateofleucine carbon (C)

ap-pearance increased (P<0.01) by 23% from 1.47±0.08 to

1.81±0.08,umol/kg- min; whereas during saline infusion leucine

C Ra didnotchange (1.50±0.08 vs. 1.57±0.09 sAmol/kg-min)

(Fig. 2). The Rd of leucine carbon also increased(P<0.01)by 24% from 1.47±0.05 to 1.83±0.09

gmol/kg

-min during the

cortisolinfusion butremained unchanged during the saline in-fusion (1.50±0.08 vs. 1.55±0.05

gmol/kg

min) (Fig. 2).

The Ra of leucine nitrogen (N) increased (P<

0.01)

by

nearly 50% from 2.35±0.12to3.46±0.24

_,mol/kg-

minduring

the cortisol infusion, but increased by only 17% (P <

_0.05)

during the saline infusion (2.43±0.17 to 2.85±0.15

Mmol/

kg.min)(Fig.3). Theincreaseinleucine N Ra

during

cortisol

administrationwasgreater(P<0.01)thanthat observed

during

salineinfusion. The Rdofleucine Nincreased

(P

<

_0.01)

_by

50% during cortisol infusion (2.35±0.12 to 3.48±0.25

umol/

kg.min) but byonly 17%(P<

0.05) during

the saline infusion

(2.43±0.17to 2.84±0.15

gmol/kg

-min)

(Fig. 3).

Effect

of cortisolon alanine metabolism. Forunexplained

reasons,plasma alanine concentrations and alanine Ra and Rd after anovernight fastweredifferent (P<

0.01)

onthetwo

days

of study. Duringthecortisol

infusion,

plasma alanine

concen-trationsdidnotchange(184±19 to 187±21 ,Mathour

8).

In

contrast, during the saline infusion plasma alanine decreased (P< 0.01)from 278±22 to 214±11

AM.

Alanine Ra

during

N =7 MEAN*SE

220

180

140

100

L

.S

2.0[

E

se

1.6

-1.2

C E. 2.0

ie

0)

1.6-E

1.2

I

..t..

o

1^°

j

or

$

.?-nifl

e1

E *p<o005 cf base line

**p<0.01 cf base line

'tp<0.05

saline vs.cortisol Vp<0.01 saline vs.cortisol *

Plasma Leu

,

l~~~~~~~~~

Ra Leu C

_-'-4,

._--.t-

t~

It t

.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~I

i I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

Rd Leu

C

I**

l

I _-~~~~~t ft --,

Ir --t

I 1I

-2 0 2 4 6 8

Hours

Figure2.Plasmaleucine(leu)concentration and Ra and Rd of leu-cine carbon(C)asestimatedby [2H3J leucine infusion inseven

nor-malvolunteersduring saline (-)orcortisol(o)infusion.

0)

-Na 7

MEANISEM

2201

180

140k

C 3.4

- 2.6

0

E

: _1.8

3.41

2.61

1.8L

.C2Kortloml0-o0

2aeg/Kg-omin o

Plasma Leu

*P<0.05 cf base line

*J..--**p 0.01 cf base line

K*

,,

-p-c0.05

saline _vscortisol If t

ltp-0.01

salinevs.cortisol '

t~~~~~~

I II

Ra Leu

N

l

li

Rd

Leu

N

P*,

I--32±12

AM).

By hour 8, the plasma alanine had decreased (P<0.01) by 64±17AM.Incontrast,duringcortisol infusion, plasma alanine did notsignificantly change (A -9±11

AM

at

hour 8, cf basal values). The change in plasma alanine concen-tration between thetwo study days was different (P< 0.05) between hour 7 and 8 (Fig. 4). Alanine Raand Rd decreased during the saline infusion but were not significantly different from basal values. In contrast, during cortisol infusion, alanine Ra and Rd were increased (P <0.05) above basal values by hour 7; and, the Ra and Rd of alanine between the infusion days were different (P <0.05) from hour 5 to the end of the study (Fig. 4).

Effect

of cortisol on leucine and alanine nitrogen interrela-tionships. The basal rate and percentage of leucineNtransfer to alanine N were greater (P<0.05) on the day of saline (0.93±0.13

Amol/kg.

min and 40±5%, respectively) when

com-pared with the day of cortisol infusion(0.70±0.03

Amol/kg

-min

and 30±2%, respectively); whereas the percentageof alanineN

N=7 MEANt SEM

40

-.2

₀

-1

-80--Ti--

-'

I a~~~~~~I

I I I I

2.0

--2 0 2 4 6 8 y

Hours

E

°F

Figure 3. Plasma leucine (leu) concentration and Ra and Rd of leu-cinenitrogen (N) insevennormal adultsduring saline (-)orcortisol

(o) infusion.

the cortisol infusion increased (P < 0.05) from 4.56±0.23 to

6.29±0.52

_gmol/kg

minby hour8,whereasduringsaline

in-fusion, itdidnotchange (5.91±0.79to5.31±0.44 ,umol/kg- min byhour8).Alanine Rd increased(P<0.05)duringthe cortisol infusion (4.57±0.24 to 6.28±0.51 umol/kg min by hour 8). During salineinfusion, alanine Rd did notchangeand values

weresimilartothose ofalanine Ra.

Since the base-linevalues forplasmaalanine concentration

weredifferenton thetwostudy days,these dataareplottedas

absolutechange from themeanbasal valuestofacilitate

com-parison between those of saline and cortisol infusion studies

(Fig. 4). Following 1 h of saline infusion, the plasma alanine

concentrations werelower(P<0.05)than thebasal values(A

< -2.0

c

E

6)

0

E

2.0

0.0

F

-2.0 L

::/

g.r

craw-or

4wfftl~e

._~~~~~

i-.r... ... ,K

Plasma Alanine

,~~

~~~~~~~*n

*

*p<.-05 cfbase line

**p<0.01 cf base line

tP<005 saline vs. cortisol lp<0.01 salinevs.cortisol

Alanine Ra

-+

Alanine

Rd

A~~~~~~~

-2 0 ~~~24 6 8

Hours

Figure4.Thechangeinplasmaalanine concentration andthe changeintherateof alanine Ra and Rd insevennormaladults dur-ingsaline(-)orcortisol(o)infusion.

415 IncreaseinProteolysisbyCortisol

c

E

6

0

E

derived fromleucine N wassimilar on the two study days ( 15±2 vs. 16±1%, respectively). Because of the differences in basal values, the dataare expressed as changefrom base line to facilitate comparison between thesalineandcortisol studies(Fig. 5).

Duringcortisol infusion, the rate of leucine Ntransferred

toalanine N was increased (P< 0.05) above the basal value during the last4hofstudy. Theincreasewas greaterover the last 3 h of study (P< 0.02) than that observedduringsaline infusion, which did not change over the course of the study. The percentage ofleucine N transferred toalanine increased (P<0.05)at hour 7 and 8 duringonly thecortisol infusion,

and wassignificantly different from thatofthesalineinfusion athour 8(Fig. 5).Anincrease (P<0.02) in the percentage of

alanine nitrogen derived from leucine wasobserved over the last 3 h during both thecortisol andsaline infusions.

Effects of cortisol infusion on the plasma concentration of

otheraminoacids. The summedtotalofall the plasmaamino

acid concentrationswasincreased (P<0.05)duringthecortisol

infusion but notduring salineinfusion(TableI). Thisisprimarily

attributable to theincreasein theplasmaconcentrationsof

va-line, leucine, isoleucine, tyrosine, phenylalanine, and histidine during cortisol but notduringsaline infusion.

Glucose kinetics. Glucosekineticsdetermined by infusion of [2-3H]glucose in threesubjects or

[3-3H]glucose

infour subjects were similarduringinfusions of cortisol or saline. As a result, these data were pooled. The small butsignificant increase (P

<0.01) in plasma glucose concentration (89±2 to 94±2 mg/dl) during cortisolinfusion was not the result of an increase in glucose Ra (2.1±0.1 to 1.9±0.1 mg/kg- min at 0 and 8 h, respectively). Therefore, the increased plasma glucose must have resulted from the relative decrease in glucose Rd asreflected

by thereduction of (P<0.02) in glucose metabolic clearance rate (2.34±0.09 to 1.96±0.16 ml/kg -min)during cortisol

in-fusion(Table II). Duringsalineinfusion, plasmaglucose

con-centrations decreased (P<0.01)from90±1 to81±2mg/dlas a result of a decrease (P < 0.01) in glucose Ra (2.2±0.2 to

2.0±0.1 mg/kg- min).

Effect

ofcortisol on plasma pyruvate, lactate, FFA, and ketone

bodyconcentrations. Plasma pyruvateconcentrationsincreased overthe course of bothstudy days, whereasplasmalactate

con-N=7

MEAN SEM

E

0

E

a

1.0

0.0

20

0

0

Be~~~~~L

W,-:-I...:: ::

...

JO ...

i~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

Leu N

--o.Ala

N

IIo

. 6 _.- . .

..II

. .

Ala

N-

LeU

N

_{a9_}

If

=*_*

wS*

5

I.

9__g__,qrTT 'Figure5. The change in therateof leucine nitrogen

trans-IlI ferredtoalanine(Leu N- AlaN), the change in

percent-ageof leucinenitrogen goingtoalaninenitrogen (LeuN

--2 0 2 4 6 8 Ala N), andthechangeinpercentageof alaninenitrogen de-rivedfromleucinenitrogen (Ala N -Leu N) in_seven

nor-Hours mal adults duringsaline(-)orcortisol (o) infusion.

*p<0.05 cf base line

I p<0.02 cf base line

tp<0.05 saline vs.cortisol

I _{p<0.02 saline}

vs.cortisol

,4

Leu N -.Aa N

*

r

1

-L

I

b.

TableI.PlasmaAminoAcidConcentrationsduringSaline

and CortisolInfusionsinNormalHumans

-60 0 240 480

Total Saline 1,692±73 1,673±78 1,623±96 1,602±80 Cortisol 1,534±119 1,466±85 1,547±102 1,694±63*

Threonine Saline 117±10 Cortisol 105±19

Serine Saline 133±3 Cortisol 122±11

Asparagine Saline 43±3 Cortisol 40±5

Glutamic acid Saline 55±3 Cortisol 59±6

Glycine Saline 272±31 Cortisol 234±26

Alanine Saline 277±24 Cortisol 193±20

Valine Saline 165±14 Cortisol 165±9

Isoleucine Saline 35±5 Cortisol 37±3

Leucine Saline 120±9 Cortisol 125±5

Tyrosine Saline 37±3 Cortisol 30±3

Phenylalanine Saline 46±6 Cortisol 40±4

Ornithine Saline 85±10 Cortisol 71±8

Lysine Saline 187±17 Cortisol 178±20

Arginine Saline 84±6 Cortisol 72±8

Histidine Saline 78±4 Cortisol 78±5 113±11 102±17 130±2 121±10 42±2 37±3 52±4 54±4 270±30 230±24 281±19 192±15 157±11 143±15 39±4 34±2 119±8 119±7 32±2 29±2 42±4 38±4 80±9 68±8 184±17 169±19 85±6 73±9 104±12 91± 15 125±4 107±10 41±5 36±3 50±4 48±5 254±27 204±25 239±14 177±16 160±10 191±14* 40±5 56±6* 132±8 167±17* 34±4 30±2 44±3 46±4 82±4 70±8 182±21 177±20 83±9 74±9 96±11 85±11 119±5 105±9 35±2 42±4 40±4 40±4 240±24 194±21 214±11 174±21 166±22 231±27* 51±6 85±8* 154±9* 203±21 *

34±3 36±2* 46±5 53±3* 86±12 72±7 184±21 183±17 77±7 78±9

79±5 81±4 82±4 76±4 82±4 85±4*

range increases the plasma leucine concentration as a result of agreater stimulation of the Ra than Rd of leucine. The increase in the leucine Ra occurred in the absence of changes in plasma insulin, C-peptide, and glucagon. Since the only source of this essentialaminoacid in thepostabsorptive state is tissue protein, weconclude that an increase of plasma cortisol within the phys-iologic range stimulates whole body proteolysis. This conclusion is consistent with the observed increased urinary nitrogen ex-cretion (14) and with elevationsin the plasmaconcentrations

of other aminoacids in this and previous studies(13-15).The tissue(s) in which this increased protein breakdown occurs cannot be determined from our studies. Pharmacologic doses of

glu-cocorticosteroidsresult inincreased3-methylhistidineexcretion (8), suggesting that proteolysis in skeletal muscle may be en-hanced. Whether a physiologic increase in plasma glucocorticoid

concentrationshassimilareffectsis not known. However, cortisol may affect proteolysis to a greater extent in tissues with more rapid protein turnover (e.g., liver, gut, skin, bone marrow) than in skeletal muscle (38).

Due totherelatively long period of isotope infusion in our study, labeled leucine may have been incorporated into and released from protein (39). This potential recycling of label would

Table II.Plasma SubstrateConcentrations and GlucoseKinetics

during CortisolorSalineInfusionsinNormalHumans

Time(hours): -2 0 4 8

Glucose

Concentration Saline 93±2 90±1 84±2* 81±2t

(mg/dO Cortisol 89±1 98±2 92±2*§ 94±2t"

Ra(mg/kg-min) Saline 2.2±0.2 2.2±0.2 2.2±0.2 2.0±0.1t

Cortisol 2.1±0.1 1.9±0.1 1.9±0.1 1.9±0.1 Rd(mg/kg min) Saline 2.2±0.2 2.2±0.2 2.1±0.1 2.0±0.1 Cortisol 2.1±0.1 2.0±0.1 1.9±0.1 1.9±0.1

Clearance Saline 2.45±0.19 2.43±0.19 2.55±0.15 2.49+0.13

(ml/kg-min) Cortisol 2.34±0.09 2.23±0.13 2.06±0.12 1.96±0.161§

Lactate (AM) Saline 940±80 840±80 670±40* 670±70* Cortisol 840±30 730±50 730±30 730±60 Pyruvate(;tM) Saline 35±11 44±12 51±14

Cortisol 46±12 55±131 60±7

Unitsaremicromolar. *_{P <0.05 from base line.}

FFA(AM)

centrations didnotchangeduringcortisol infusion but decreased

during saline infusion (P < _0.05) (Table II). Plasma FFA,

f3-hydroxybutyrate,

and acetoacetate concentrations increased

similarlyon bothstudydays (Table II).

82±10*

85±5t

Saline 810±120 830±100 890±70 1060±90t

Cortisol 530±100 650±60 830±90 990±1101 0-Hydroxybutyrate Saline 200±100 320±100 410±110* 870±160* (AM) Cortisol 210±41 280±46 600±100t 1070±140t

Acetoacetate(pM) Saline 150±50 170±50 220±50* 420±50t

Cortisol 120±20 160±30 320±30t 470±40t

Totalketone bodies Saline 450±160 490±150 720+154t 1300±210t

(puM) Cortisol 330±60 440±66 910±100t 1540±140t

Discussion

Thepresentstudy, using isotope dilutiontechniques,

demon-stratesthatanincrease inplasmacortisol within thephysiologic

*_{P<0.05cf baseline.}

tP<0.01cf base line. §P<0.05 cfbaseline. 11P<0.01 cf saline. IP<0.02cfbaseline.

resultin anunderestimation in the Ra ofleucineduringboth saline and cortisol infusions. Therefore, it ispossiblethat the cortisol-induced increase in proteolysis observed at 3 h may have occurred evenearlier and/or toagreaterextent.Thus, our calculations may representanunderestimation of theeffect of cortisol onproteolysis inman.

In addition to an increase in the Ra ofleucine, cortisol infusion increased the Rd of leucine. Leucine released from

body proteincanbeeither utilizedforprotein

synthesis

or

un-dergo irreversible oxidative decarboxylation following

trans-aminationtoitsa-ketoacid, a-ketoisocaproate (KIC). Highdose in vivo glucocorticoid treatment inhibits protein synthesis in rat skeletal muscle (9-12), butits effect on nonskeletal muscle protein is not known. Ifglucocorticoidsnotonlystimulate pro-teolysis, as the present studies demonstrate, but also inhibit

proteinsynthesis, then therateof leucine oxidationwould have toincreasedramatically. Thiswould leadtoaccelerated loss of

an essential amino acid and ultimately to severe protein de-pletion. Althoughglucocorticoidexcessresults innegative protein

balance, major protein lossoccursonly after weeksofexposure

topharmacologiclevels. Therefore,theincreaseinleucine

uti-lizationmayreflectsomestimulation of leucineoxidation, but mostlikely represents anincreaseinproteinsynthesisin some body tissuesaswell.Clarification of the ultimate fate of leucine releasedfrom bodyproteinawaitsthemeasurementof leucine

oxidation undersimilarexperimental conditions.

Asdemonstrated here and in previous studiesinman and dogs(16-18,40), theflux of leucine nitrogen,asestimated from

the

["N]ileucine

data, exceeds that ofleucine carbon. Since

[12H3]1eucine

is

probably

transaminated to KIC and back to

leucine withoutlossoflabel whereas the '5N would be lost to

alargenitrogenpool, these resultsareconsistent witharateof

leucine transamination thatis higher than irreversible loss of

leucine carbon. Whetherthishighrateof transamination reflects "futile" transamination ofleucine inasingletissueorits

trans-port as the a-ketoacid from peripheral tissues to liver under theseconditions remainsunknown;however,recentstudies in the rats (41) and dogs (42) lend support to the latter hypothesis. If the ratioofleucine N toleucine Cflux is areflectionof the

activity ofthe proposedleucine-KIC shuttle, then cortisol has noeffectontherelative activity of this shuttle since the changes in the ratio of the leucine N/leucine C flux were identical during

cortisol and salineinfusions(1.47-1.80 withsaline; 1.47-1.89 with cortisol).

In the postabsorptive state, alanine appearance into the plasma space represents the sum of alanine release from protein (proteolysis) and production from de novo synthesis (16-18, 43). Assuming alanine and leucine content of body protein are

6 and 8%, respectively (44), the rate of alanine release from protein can be calculated from leucine C appearance rates (16-18, 43). The calculated rateof alanine release from protein

during cortisol infusion increased23%(from1.63 to 2.00

,umol/

kg min), whereas during salineinfusion, it increased by only 5% (from 1.66 to 1.74

,umol/kg.

min). The total alanine Ra

increased from 4.56 to 6.29

_gmol/kg-

min during cortisol in-fusion; therefore, the rate of alanine synthesis (alanine Ra

- alanine released from protein) increased by 46% from 2.93 to4.29

_,mol/kg

* min. In contrast, alaninesynthesis during saline infusion did not increase (from 4.25 to 3.57 ,mol/kg * min). The fact that the rate and percentage of leucine N going to alaninesignificantly increased only during cortisol infusion is furtherevidence ofacortisol-mediated stimulation of de novo alanine synthesis. Because the increases in the percentage of alanineNderived fromleucine were notdifferentonthetwo

study days, we conclude thatcortisol didnotselectivelyaffect thetransferofNfromthebranched-chainamino acids to alanine. Itis of interest to note that the difference in basal alanine flux

onthetwostudydays is attributabletodifferences in the rates ofalaninesynthesis, rather than ofproteolysis,since basal leucine carbonratesof appearanceweresimilaronthetwostudydays. This interpretation is supported by our observation of higher basal rates and percentages of leucine N transfer to alanine during the base-lineperiod of study on the saline infusion day

ascompared with theday of cortisol infusion.

During cortisol infusion alanine Ra increased. However,

alanine concentrationdid notchange because its Rd increased tothe same extent. In contrast, the plasma alanine concentration during saline infusion decreased. Thiswaspresumablydueto adecrease inalanine Ra, sincealanine Rddidnotincrease. It should be noted that the changes in the plasmaconcentration

of lactate, a potential carbonsourceforalaninesynthesis, par-alleled those ofalanineonthetwostudydays. However, plasma concentrations of pyruvate, the immediate precursortoalanine synthesis, didnotfollowthis pattern. Duringcortisolinfusion,

the sustained plasma lactate and alanine concentrations and the increase in alanine turnover were associated with higher

glucose concentrations than observed on the control day.

Whetherthishigher plasma glucoseconcentration was the result of increasedavailabilityof thesegluconeogenicsubstratescannot

be determinedfrom the presentstudies,sinceacarbon-labeled alaninewas notused.

Insummary,the presentstudies demonstrate thatanincrease ofcortisol within the physiologicrange stimulates proteolysis independentlyof changes in plasmainsulinandglucagon. The

increasein alanine turnover observed during cortisol infusion was notonly the resultof enhanced proteolysis but of an even greater increase in alanine synthesis. The latter is consistent

with acortisol-mediated stimulation of the postulated glucose-alanine cycle (45). The increasedavailability ofpotential

glu-coneogenicsubstrates(alanine andpossiblylactate and pyruvate) maycontribute to the changes in carbohydrate metabolism ob-servedduring stressand otherhypercortisolemicstates.

Acknowledgments

This workwassupportedby the WasieFoundation,MayoFoundation,

andNationalInstitutes ofHealth grant AM-26989 and AM-2041 1,

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