Combined and

Combined Pancreas and Kidney Transplantation Normalizes Protein Metabolism in Insulin-dependent Diabetic-Uremic Patients

Livio Luzi,*AlbertoBattezzati,* GianlucaPerseghin,* EldaBianchi, * IleanaTerruzzi,* DonatellaSpotti,* SandroVergani,t Antonio Secchi, * Ennio La Rocca,* GiovanniFerrari, CarloStaudacher, Renato

Castoldi,I

Valerio DiCarlo,#and Guido Pozza*

Departments of*InternalMedicine,ISurgery,and*0phthalmology,SanRaphael ScientificInstitute,University ofMilan, 20132 Milan, Italy

Abstract

In order to assess thecombined and separate effectsofpan- creasandkidney transplantonwhole-bodyproteinmetabolism, 9insulin-dependentdiabetic-uremicpatients(IDDUP),14pa- tients after combined kidney-pancreas transplantation (KP- Tx), and 6 insulin-dependent diabetic patients with isolated kidney transplant (K-Tx), werestudied in the basal postab- sorptive stateandduringeuglycemichyperinsulinemia (study 1).

11-14ClLeucine

infusion and indirectcalorimetrywereuti- lized to assessleucine metabolism. Thesubjectswerestudied again withacombined infusion of insulin and aminoacids, given tomimicpostprandial amino acid levels(study 2).Inthe basal state, IDDUP demonstrated withrespect to normal subjects (CON): (a) higher free-insulin concentration (17.8±2.8 vs.

6.8±1.1

AU/ml,

P< 0.01) (107±17 vs. 41±7

pM); (b)

re- duced plasma leucine (92±9 vs. 124±2

MM,

P < 0.05), branchedchain amino acids(BCAA) (297±34vs.416±10

AM,

P < 0.05), endogenous leucine flux (ELF) (28.7±0.8 vs.

39.5±0.7 Mmol*m-2 *

min-',

P<

0.01)

and nonoxidativeleu- cine disposal (NOLD) (20.7±0.2 vs. 32.0±0.7 Mmol.m2-

min-',

P<0.01); (c)similarleucine oxidation

(LO) (8.0±0.1

vs.7.5±0.1 Mmol*m-2

min';

P= NS).^BothKP-TxandK- Txpatientsshowed acomplete normalization ofplasmaleucine (116±5 and 107±9 MM), ELF (38.1±0.1 ^and 38.5±0.9

Amol

-m-2.min-), and NOLD (28.3±0.6 and 31.0±1.3 Amol*m-2.

min-')

(P= NS vs.CON). Duringhyperinsulin- emia(study 1),IDDUPshowedadefective decrease of leucine (42%vs.53%;P<0.05), BCAA(38%vs.47%,P<0.05),ELF (28%vs.33%,P<0.05), and LO (0%vs.32%,P <0.05) with respect toCON. Isolatedkidney transplantrevertedthedefec- tiveinhibition ofELF(34%,P=NSvs.CON)ofIDDUP, but nottheinhibitionof LO(18%,P<0.05vs.CON)byinsulin.

Combinedkidneyand pancreastransplantationnormalizedall kineticparametersof insulin-mediated protein turnover.Dur- ing combined hyperinsulinemia and hyperaminoacidemia (study 2), IDDUP showeda defective stimulation of NOLD (27.9±0.7vs.36.1±0.8Amol m-2.

min',

P <0.01compared

AddresscorrespondencetoDr.Livio Luzi at his current address: Sec- tion of Diabetes and Metabolism, Division of Endocrinology-Hyper- tension, Brigham and Women's Hospital, 221 Longwood Avenue, Boston,MA02115.

Receivedforpublication 2 April 1993 and inrevisedform 8 October 1993.

toCON), whichwasnormalized by transplantation(44.3±0.8 Mmol-m2

min'). (J.

Clin. Invest. 1994. 93:1948-1958.) Keywords: diabetes mellitus * uremia* kidney transplant*kid- ney-pancreas transplant * protein turnover

Introduction

Both diabetes mellitus and chronic uremia determine pro- found alterations ofwhole-bodyproteinmetabolism(1-3).In thefastingstate,poorlycontrolleddiabeticpatientsarecharac- terizedby negative nitrogen balance, increased blood amino acidconcentrations, and increased proteinturnoverand oxida- tion rates, as assessed bycommonly employed isotopetech- niques (4-6)^.In contrast,patientswith chronic uremiausually presentreducedbloodamino acid concentrations and reduced whole-bodyleucineturnoverandoxidationrates(7).There- sult of these metabolic alterations isa negative nitrogenbal- ance. Furthermore, intheinsulin-stimulated condition,both diabetes mellitus(4) and chronic uremia (8)arecharacterized by insulin resistance with respect to glucose metabolism, whereas the insulin effect onprotein metabolism is normal.

Thissuggeststhat thedefect in insulin action isatpostreceptor- ialsites in both diseases.

Isolatedkidneyandcombinedkidney-pancreastransplan- tation constitute validtherapeuticapproachesinthetreatment ofinsulin-dependentdiabetic patientswhodevelop end-stage renal failure. Previousreportsalreadyemphasizedtheimpor- tanceofco-transplantingthepancreasalongwith thekidney,in ordertoobtain a near normalization of glucose metabolism (9-15). The effect of kidney-pancreas transplantation on whole-body protein metabolism is still unknown. An addi- tionalproblem is constituted by the needtoadminister chronic immunosuppressive therapy in diabetic patients after kidney andpancreas transplantation. The specific aims of thiswork are (a) to assessthe effect of isolated kidney transplantand combined kidney-pancreas transplantation on basal leucine turnoverand oxidation rates(whichare an estimate ofboth protein catabolism and protein synthesis), (b) to assess the effect ofacutehyperinsulinemiaonleucine/protein metabo- lism before and after isolatedkidney and combined kidney- pancreastransplantation,(c) to study theeffectofcombined hyperinsulinemia/hyperaminoacidemia on protein synthesis before and afterkidney-pancreastransplantation,(d)todeter- mine theeffect of chronic immunosuppressivetherapyper se on leucine/protein metabolism in patients with posterior chronic uveitis(localized ocular diseasewithout systemic dis- ease).Our results demonstrate thatcombinedkidney-pancreas transplantation completely reverts the alterations of amino acid/proteinmetabolismindiabetic-uremicpatients.

1948 Luzietal.

J.Clin. Invest.

©TheAmericanSociety forClinicalInvestigation, Inc.

0021-9738/94/05/1948/11 $2.00 Volume 93, May 1994, 1948-1958

Table I. Clinical and Laboratory Data ofthe Five StudyGroups

GroupI Group 2 Group3 Group 4 Group 5

(IDDUP) (KP-Tx) (K-Tx) (CU) (CON)

Number 9 14 6 5 8

Age (yr) 35±4 36±2 34±5 31±4 32±5

Duration of diabetes(yr) 20±4 22±3 19±4

Bloodpressure(mmHg) 166±3/97±2* 148±3/87±1 150±5/88±6 128±2/84±3 121±4/82±3

Blood pH (pH units) 7.32±0.04 7.38±0.02 7.36±0.03 7.40±0.02 7.40±0.02

Plasma glucose(mg/dl) 133.4±9.0011 95.4±7.2 118.8±3.6*1 86.4±1.4 89.3±2.1

Insulin dose(U/d) 44±3 48±4

HbA1C(%) 9.2±0.3*1 5.9±0.2 8.7±0.4*11 5.6±0.2 5.2±0.1

Plasma urea(mg/dl) 86.8±23.5* 21.5±0.5 34.7±0.3 24.3±2.5 15.6±0.2

Plasma creatinine(mg/dl) 10.2±1.1t 1.1±0.2 1.3±0.1 1.1±0.1 0.8±0.2

Nitrogen excretion(mg/min) 11.5±0.8* 7.0±0.2 8.4±0.7 8.0±0.3 8.1±0.5

Plasmafree-insulin(MU/ml) 17.8±2.8*1' 14.8±1.6*11 6.2±1.8 9.1±1.0 6.8±1.1

Plasma C-peptide(ng/ml) 0.06±0.01*1 2.89±0.32* 0.05±0.02511 2.51±0.14* 1.70±0.20

Plasma growth hormone(ng/ml) 8.93±2.12*111 2.02±0.30* 1.35±0.16* 0.47±0.14 0.51±0.07

Plasma glucagon(pg/ml) 152±24* 127±19 162±7* 120±14 68±7

Plasma cortisol(ng/ml) 154+170§11" 65±14 52±2 28±4* 71±7

Prednisone dose(mg/d) 10±2 10±1 15±3

Cyclosporinedose(mg/kg perd) 5±1 6±1 5±2

Azathioprinedose(mg/kg perd) 1.0±0.2 1.1±0.1 1.1±0.1

Conversion factorstoSIunits: glucose = 0.05551, urea=0.357, creatinine=88.4, free-insulin=6.0, C-peptide=0.331, growth hormone= 1.0, glucagon= 1.0,cortisol=2.759.

*P<0.05 vs. CON;*P <

0.01

vs.CON;

lP

<

0.01

vs. KP-Tx;"P <

0.01

vs.CU;'P<

0.01

vs.K-Tx.

Methods

Subjects and experimental protocol

9insulin-dependent diabetic-uremic patients (IDDUP';bodymassin- dex[BMI]22.1±2.0; five males, four females), 14patients aftercom-

bined kidney-pancreas transplantation (KP-Tx;BMI22.1±2.4;eight males, sixfemales), 6insulin-dependent diabetic patients after isolated kidney transplant (K-Tx;BMI22.3±2.1;three males, threefemales),5 patientswithchronic uveitis (CU)onthesameimmunosuppressive therapyastransplantedpatients (BMI 23.0±2.2;threemales,twofe- males), and 8 normal healthy controls (CON; BMI 23.8+2.9; five males, threefemales)werestudied.All transplantswerefromcadaveric donors. 8 patients receivedasegmentaland 6 patients receivedatotal

pancreastransplant. The patientswereadmittedtotheMetabolic Unit ofSanRaffaeleHospitalat8:00a.m.,atleast 24 hbefore the study. All subjects underwent a 180-min euglycemic hyperinsulinemic (40 mU m-2 *min')clamp, combinedwith indirectcalorimetryasprevi- ouslydescribed(13).AllIDDUP,9patientsafterKP-Tx, and all K-Tx, CU,andCONsubjects underwenta[I-'4C]leucine infusiontoassess

whole-body leucineturnoverandoxidation.Inthe remaining KP-Tx patients(5)aeuglycemic insulinclampwithouttracerinfusionwas

performed.Therefore, onlydataoncold substrateconcentrationare

available forthosesubjects.Allthesubjectsfollowedanisocaloric diet containingatleast250gofcarbohydratesand70-90gofproteinsper

dayin the 2 wkpreceding thestudy. Theday before the study the fat-freemass wasassessed in allsubjects bymeansofwhole-bodybio-

1. Abbreviations used in thispaper: BCAA, branched chain amino acids; BMI, body massindex; CON, normal subjects; CU, chronic uveitis(patients); ELF, endogenousleucineflux; IDDUP,insulin-de-

pendent diabetic uremic patients; K-Tx, kidney-transplanted (pa- tients); similarly, KP-Tx,kidney/pancreas-transplanted (patients);^a- KIC, a-ketoisocaproic acid; LO,leucineoxidation; NLB,net leucine balance; NOLD,nonoxidative leucinedisposal.

electricalimpedance aspreviously described ( 16, 17). In IDDUP the fat-freemass wasassessedatthe end ofthe hemodialytic session( 17).

IDDUPweredialyzing 3 d/wk; the study wasusually done within 48 h from the lasthemodialytic session. All subjectswerefully informed of thepossible risks ofthestudy and gave theirconsent.The experimental protocolwasapprovedby the Institutional Ethical Committee. Table I summarizesthe clinical and laboratory data of the fivestudy groups.

Allvalues in the table refer to bloodsamples drawn themorning of study 1.

Afteranovernightfast,at6:00a.m. apolyethylene catheter ( 18G, Terumo, Leuven, Belgium) was inserted intoanantecubital veinfor the infusion of alltestsubstances. Asecond catheterwasinserted retro- gradely intoawrist vein andadvancedtothe dorsum of the hand for blood sampling. The hand wasplaced in an heated box (70'C)to ensurearterialization of thevenousblood( 18).Aprime-continuous infusion of[1-_4C]leucinewasadministered( 16gCias abolus injec- tion, followed by 0.20MCi/minas acontinuousinfusion; Amersham Corp., Arlington Heights, IL; aqueous solution containing 2% eth- anol), in combination withapriming dose ofNaH'4C03 (2.3ACi, Amersham, sodium salt)toprime thebody bicarbonate pool (4, 19).

After2hofisotopeequilibration,samplesweredrawnat10-min inter- valsfrom 120 to 180 min for the determination of basal leucine and a-ketoisocaproic acid (a-KIC) specific activities,aswellashormone andsubstrate concentrations.

Continuous indirectcalorimetrywasstarted 90min after thetracer infusionwasinitiated,and continuedthroughoutthestudy.Expiredair sampleswerecollectedat15-min intervals and bubbledthroughaCO2 trappingsolution(hyaminehydroxide/absolute ethanol/0.1% phenol- phthalein, 3:5:1) asdescribed previously (4, 19). The solutionwas titratedtotrap 1 mmolCO2per 3 ml of solution. The'4C radioactivity wassubsequentlydetermined witha#scintillationcounter(Packard InstrumentCo.Inc.,Meriden, CT)and theexpired '4CO2 specificactiv- itywascalculated.Total14CO2expiredper minutewasdeterminedby multiplyingthe '4C02 specificactivity bythe totalCO2 production, determinedasdescribed below. Theexperimentalprotocol is depicted inFig. 1.

Study 1: insulin clamp. Allthesubjects participated in thisstudy protocol. After a 180-min equilibration periodaninsulin clampwas

performed as previously described (13). Briefly aprime-continuous infusion of crystalline human insulin (ActrapidHM, NovoNordisk, Copenhagen, Denmark) was administered at the rate of 40 mU_m-2.min'toachieve and maintainanincrement in plasmain- sulin concentration of ^- 75MU/ml. The plasma glucose concentration was maintainedat thebasal level by measuringitat5-minintervals and by periodic adjustment ofa20% glucose infusionbasedon anegative feedback principle.

Study 2:insulin clamp withaminoacid infusion. SixIDDUP,seven

KP-Tx patients, fourK-Txpatients, fourCUpatients,andfiveCON were studiedwitha repeatinsulinclamp. Theinsulin clampwas per-

formed as described above withoneexception.Abalancedaminoacid solution was begunatthestartof the insulin infusionandcontinued throughout thestudyaspreviouslydescribed (4). The rateofamino acid infusion averaged 0.98±0.11 ml*m_2.min'(cold leucineinfu- sion rate of51±6

MmolI* m-2.

min-')in all study groups. These infu- sion rateswere chosentosimulatethepostprandialplasmaaminoacid

STUDY I

120

I

.5, 60

01

Indirect calorimetry

(1-"'C]

^leucine

150

100

50

-1800 lO0

-180 0IS

STUDY 2

Z Amino acids

I Insulin

I

Indirect calorimetry <

[

1-"C]

^leucine

120-

60F

0 -180

100

40 0

Figure 1. Experimental protocolsof both Studies1 and 2. [I-"'C]-

Leucine infusion andindirect calorimetrywereperformedin both studies. Duringstudy 1, after 180min of basal equilibration,a40 mU* m-2 -min'insulin infusionwasstarted and continued for 180

min.

Study 2wassimilartostudyI withoneexception.Abalanced amino acidsolutionwasinfused along with insulin inordertodouble the plasma amino acidconcentration. Solidlines refertoplasmaleu- cine;dashedlines refertoplasma glucose level.

(vn)

^{IDDUP;(,&)KP-}

Tx;(A&)K-Tx;(o)CU;(e)CON. 'P<0.01 vs.basal; *P<0.01 vs.

groups2,4, and 5.

concentrations.The aimwas toobtainthesameincrementwithrespect to thebasal levels in all groups.

Respiratoryexchangemeasurements.Inallstudies,totalCO2pro- ductionrateand 02consumptionrate weremeasuredbycontinuous indirectcalorimetryasreportedpreviously(13), bymeansof Sensor- Medics instrumentation, ModelHorizon,Anaheim, CA.Nonprotein respiratory quotientwasmeasuredusingthe Lusk's tableasreviewed anddiscussed by Simonsonand DeFronzo(20). Nitrogenexcretion ratein the IDDUP wasquantifiedaspreviously described (13).

Analytical determinations and calculations

Plasmaleucine concentration and specific activityweredetermined using an HPLC system (System Gold, Beckman Instruments, Inc., PaloAlto,CA) constituted byaprogrammablesolvent module(model 126AA) and a programmable detectormodule, model 168 asprevi- ouslydescribed (4). Briefly,toprecipitate plasmaproteins 50 mg of

sulfosalycilic

acid(drypowder)wasadded to 1.0 mlof plasmaanda

100-Al

aliquot ofthe supernatant wasanalyzed induplicate for amino acid concentration. 1 ml of theremainingsupernatant was placed in duplicate on acation exchangeresincolumn (Dowex 50G,Bio-Rad Laboratories, Richmond, CA) and the free amino acidfractionwas elutedwith 4 NNH40H,dehydrated,andreconstitutedin water. 10 ml ofPicofluor 15scintillation fluid (Packard)wasaddedtoeachvial and

'4C

radioactivity was measured in ascintillationcounter.The recovery of

[1-'4C]leucine

added to plasma was98±2%. Theinterassay and intraassayvariations forthe determination of[1-"4C]leucine specific activitywere3±1% and 4±2%,respectively. > 98%oftheradioactivity collected in theaminoacidfractionwasinthe leucinepeakafter separa- tion by ionexchange chromatography (21). Plasma a-KIC specific activity was measured as previously described (4, 22).Briefly, 1 ml of plasma was placed in duplicate on a Dowex 50 G cation exchange resin column (Bio-Rad Laboratories) and the free a-ketoacid fraction was elutedwith4 mlof 0.01 NHClin 50 ml cell culture tubes. 35 mlof methylene chloride was added and after shaking vigorously for1 min the tubes werecentrifuged for5 min at2,000 rpm to extract thefree a-ketoacid fraction from the plasma. After decanting the supernatant the a-ketoacids were extracted in 350Ml of 0.2 M NaH3PO3 at pH 7.0.

After a brief centrifugation 200 Mlof the resultant supernatant was injected into an HPLC system. The system utilized aCl8 reverse-phase column (Altex-Ultrasphere ODS, 0.6X25cm; Beckman,Instruments, Inc., Palo Alto, CA) that was eluted with 5% acetonitrile in 0.2 M H3P04buffer (pH 7.4) at the rate of 1.4ml/min.Absorbance of a-KIC was monitored at 206 nm. Radioactivity eluting with the a-KICpeak was measured by liquid scintillation

#-counting

^{(4). All}calculations were made during the last hour of the tracer equilibrationandof the insulin clamp period when steady-state conditions for leucine and a- KIC specific activities and concentrations existed. Data were analyzed using a stochastic model as previously described (4, 23). Themodel generates the following equations in which the total leucineturnoveror flux equals: Q=S + C=B +I,where Sisthe rate of leucineincorpora- tion into protein (nonoxidative leucine disposal); C is the rate ofleu- cine oxidation; B is the rate of leucine release from protein (endoge- nous leucine appearance); and I is the rate of exogenous leucineinput.

The rate of leucine turnover ( Q) is calculated as follows: Q =F/leucine specific activity, where Fisthe infusion rate of[1-"4C]leucine (indpm/

min)and

leu

specific activity is the specific radioactivity ofleucinein the plasma compartment under conditions of equilibrium. Theleucine oxidation rate (C) is calculated as follows: C=O/(K- leucinespecific activity), where 0 is the rate of appearanceof "CO2in theexpired air (in dpm/min) and K is a correction factor (0.81) which takesinto account the incomplete recovery of labeled "CO2 from thebicarbonate pool. In particular, the 0.81 factor accounts for thefractionof"C02 released into the body's bicarbonate pool from the oxidation of[1-"4C]- leucine, but not recovered in expired air (24, 25). Anestimateofthe rate of leucine incorporation into protein (S) can be calculated as follows: S= Q- C. An estimate of the rate of leucine release into

plasma space

fromendogenous protein(B)canbecalculatedas fol- lows: B= Q-I.

1950 Luzietal.

--$-*~~ ---- ~~

^T

1

la

I E

A

C

When thesubjects are in the postabsorptive state, the leucine intake (I) equals0and B = Q. When amino acids are being infused intrave- nously, the rateof exogenous leucine infusion (I) must be subtracted from the total leucine flux (Q) in order to calculate the rate of endoge- nousleucine release from proteins. To calculate the rates of leucine turnover andoxidation, we used the plasma leucine specific activity.

Recentpublications have suggested that the plasma specific activity of thetransaminated product of leucine, thea-KIC, may provide a better indicator of the specific activity in theintracellularmixing pool (26, 27). Therefore, estimates ofleucine kinetics were also carried out using the plasma a-KIC specific activity.

Plasma glucose concentrations were determined by a differential- pH method previously described (28). Methods for determining plasmafreeinsulin ( 13), glucagon ( 13), cortisol ( 13), growth hor- mone (13), andepinephrine (29) concentrations have been previously described. Nitrogen excretion rate was assessed as previously de- scribed ( 1 3 ).

Statisticalanalysis

Allvalues are expressed as the mean±SE. Comparisons between the basal and theinsulin-stimulated states within a group were performed with the Student's t test for paired data. Comparisons among the various groups wereperformed with the ANOVA and the Student'st test(30).

Results

Plasma glucose, hemoglobin (Hb) Ac, hormone concentra- tions, and nitrogenexcretion rate (TablesI-III). In the basal state,plasma glucoseconcentrationwashigher inIDDUP and K-Txrespect to KP-Tx, CU, and CON (P <0.01).In contrast, during the last hour of studies 1 and 2,it didnotdiffersignifi- cantlyamongthevarious studygroups(TablesI andIIIand Fig. 1). HbAlC concentration was increased in IDDUP and K-Txwithrespect tothe other groups (Table I). Postabsorptive free-IRI concentrationwasincreased inthe basal state in ID- DUPand KP-Tx with respect to CU and CON (P < 0.01).

Duringstudies1 and 2 theincrementof free-insulinwassimi- lar in thefivegroups(TableII,Fig. 1). Basal glucagonconcen- trationwashigherin IDDUP andK-Txwithrespect toKP-Tx, CU, and CON (P<0.05) (Table I).Duringbothstudies1 and 2, plasma glucagonconcentrationdidnotchangesignificantly from thebasal state, in any of the groups(Table II). Basal growth hormone concentration was increased(P< 0.01) in IDDUP(8.93±2.12)vs. KP-Tx(2.02+0.30),K-Tx(1.35+0.16), CU (0.47±0.14), and CON (0.51±0.07). Growth hormone concentration increasedsignificantly with respectto basalin KP-TxandK-Tx(P<0.05)duringeuglycemic hyperinsulin- emia(Table II). Cortisol concentrationwashigherinIDDUP vs.allgroups(P<0.01) (Table II). Plasma epinephrinecon- centration was increased in the basal state in IDDUP (1,961±320 pM)vs.allgroups(P<0.01) (TableII).

In the basal state, plasma FFA were similar in IDDUP (0.589±0.08),KP-Tx(0.521±0.06),K-Tx(0.681±0.04),CU (0.610±0.01), and CON (0.592±0.01 mM) (Table III).Dur- ingeuglycemic hyperinsulinemia the FFA concentration de- creasedofasimilarvaluein IDDUP(0.398±0.01)respectto KP-Tx(0.358±0.01), K-Tx (0.524±0.02), CU(0.457±0.1), andCON(0.449±0.2 mM).

Thenitrogen excretionrate wasincreasedin IDDUPwith respect toKP-Tx, K-Tx,CU,and CON(P<0.01).

Fat-freemass. InIDDUP(BW=62.1±3.2kg)thefat-free mass (73.7+2.9%) was slightly reduced compared to KP-Tx

(77.2±2.1 %),K-Tx(74.2±1.8%),CU(74.4±2.1 %), and CON

(75.4±2.3%). However, no statistically significant difference was detectedamong any of the groups.

Plasmaamino acid, leucine, and a-KIC concentrations and

specific

activities in thepostabsorptive state in the

five

study groups (Table III, Fig. 1). In IDDUP, the fasting branched chainamino acid(BCAA) (297±34

,M),

andleucineconcen- trations (92±9

gM)

werealllower(P<0.05)than those in controlsubjects (CON: 416± 10 and 124±2 MM for BCAA and leucine, respectively)(Table II). Thefastinga-KIC concentra- tion(19±3 vs. 28±2 AM) was also lower (P < 0.05) with re- spect toCON.AfterKP-Txand ininsulin-dependent diabetic patients after isolated K-Tx, the basal plasmaBCAA(340±14, and 311±11 uM, respectively) increased with respect to ID- DUP,still being significantly lower than normals(P < 0.05).

BasalBCAA inCU (459±321M)wassimilartothoseinCON (416±10 AM). Basal plasma leucine (116±5, 107±9, and 121±8

,M),

and basal plasmaa-KIC(23+1, 24+2,and35±4

uMM)

in

KP-Tx, K-Tx,

and

CU, respectively,

didnotdiffer from those in CON (Table II). Basal plasma leucine, (3.61±0.52 dpm/nmol) and a-KIC (2.44±0.71 dpm/nmol) specificactivi- ties inIDDUP weresignificantly highercompared withthose of CON(1.94±0.21 and 1.27±0.10dpm/nmol, respectively;P

< 0.05), as well as in KP-Tx, K-Tx, and CU, respectively (plasmaleucine [2.93±0.24,

2.63±0.48,

and2.91+0.03dpm/

nmol] and a-KIC [1.97±0.11, 1.77±0.22, and 1.79±0.08 dpm/nmol]). Plasma phenylalanine concentration was not statisticallydifferentamongthefivestudygroups(Table III).

Plasmaalanine concentration washigherinIDDUP andCU vs. Groups KP-Tx, K-Tx, andCON subjects(P <0.05). A steady-stateplasma plateau wasachieved for the leucine and a-KICspecific activities during the last hour oftheequilibra- tionperiodin allstudygroups.

Plasmaaminoacid, leucine,anda-KICconcentration and

specific

activitiesduring studyI(hyperinsulinemia) andstudy 2 (combinedhyperinsulinemia/hyperaminoacidemia) (Table III,Fig.1). During study 1,IDDUPdemonstratedadecrease in all measured plasma amino acid concentrations with the notable exception of alanine. The BCAA concentration de- creasedfrom 297±34to

182+25,

from 340±14 to

151+9,

from 311±11 to

133+24,

from 459±32 to 264±28, and from 416± 10 to221±13 AM in IDDUP, KP-Tx, K-Tx, CU, and CON, respectively. Plasma leucine concentration decreased from 92±9 to 53±6, from 116±5 to 42±5, from 107±9 to 43±4, from 121±8to59±4, andfrom 124±2 to58±2,Min IDDUP, KP-Tx, K-Tx, CU, and CON,respectively. Plasma a-KIC concentration decreased from 19±3 to 12±2, from 23±1 to 14±1,from24±2 to 12±1, from 35±4to 19±2,and from 28±2 to 14±2 ,uM inIDDUP, KP-Tx, K-Tx, CU, and CON, respectively. The absolute decrease ofplasma leucine concentration from baseline(39±6 vs.66±4

,M)

wassignifi- cantly less(P< 0.01)in IDDUPcomparedtoCON

(Table

III).Inthepatientsstudied after KP-Tx, the absolute decreases in BCAA(189±36vs. 195±20,uM),leucine(74±11 vs.66±4 MM), and a-KIC (9±1 vs. 14±2 MM) concentrations were

nearlyidenticaltothoseobserved in CON(Fig.1,TableIII).A steady-stateplateau ofplasmaleucine and a-KICconcentra-

tionsand specificactivitieswasachievedin allstudy subjects duringthe last hourof insulin infusion.

The plasma BCAA (487+58, 640±84, 598±92, 602±87, and

683±69,MM),

leucine(157±22,220±8,

207±23,

209±

17,

and 215+5 uM), and a-KIC(23+2, 23+1, 29+2, 30±3,and

27±3,MM)

concentrationsweresimilar inIDDUP,KP-Tx,K-

+1 +1

N -

N0 0 +1 +1

00 0

+1 +1 00 00

0n

+1 +1

00 00

00 N 0 +1 +1

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+1 +1

00 ;

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6A"i

+1 +1

N- ON

00

N- en +1 +1

+1 +I

00 I'D

0q

+1 +1 +1 +1 +1

+1 +1 +1 +1 +1

9D

+1 +1 +1 +1 +1

0 ~ cN N, kn

0 cq'I

-

rm 0

lf 6

+1 +1

Nl 0

-o 0

+1 +1

r--:

Nl

+1 +1

0

aN

+1 +1

a-, -7 (N!

0%1

+1 0

00

+1

(N4 +1

ON

+1

* *~~~~4

el e(N en)

+1 +1 +1 +1 +1

Oo

0L

Cu

0.0

0

C;V

oO

(N>

~2V

0-)Z

aw

0U

+C

05

0_

+E

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0_

0% 00

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00

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0 0 6 0

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a,% 4

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0D 00 0D 1

I0

T4 0 (N m

+1 +1 +1 +1 +1

:t 00 0 - 00

0

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0 00 +1 (N4 r(4

00 00

T- o

+1 +1 +1 +1 +1

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00

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a, m

00

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41+1

+1 +1

:t 00

+1 +1

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+1 +1

(6 +1

+1

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+1 0D

+1 N- 00

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00

+1

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+1 +1

'c a's

%0o N-

00 +1

+1 N-

4+1

00 0%

4

+1 +1 +1 +1 +1 +1

nr 00 00

*N *en

+t +1 +1 +1 +1 +1

(N4 ON N-

ON*_rr

en +1

+14

+1 ON

(N

+1

0

2

~~CO

co 00 Cc -~c

WI w CO

a..

0

0.

>

Cu-

<V

CA.

0v0

,ui

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0i

1952 Luzietal.

+E

0 (A

+S

0

+S (A

0E

06Z

enH

_OC

a:)0.

=a

2a 0--

z

C..,

C..

C..)

C.)

C..

C..,

C-)

C..

C.)

C..C..

k

Tx,CU,andCON duringthe lasthourof study2.Theplasma leucine anda-KICspecific activities,respectively,weresignifi- cantly higher (P<0.05) in IDDUP (1.71 ±0.11 and1.66±0.19 dpm/nmol)with respecttoKP-Tx(1.46±0.17 and 1.31±0.14 dpm/nmol), K-Tx (1.33±0.31 and 0.96±0.11 dpm/nmol), CU (1.34±0.28 and 0.88±0.23 dpm/nmol), and CON (1.34±0.12and 1.09±0.09dpm/nmol).

Total

leucineflux.

Inthe

postabsorptive

state,total leucine fluxaveraged28.7±0.8 Omol*m2. min'-inIDDUP,a

signifi-

cantlylowervalue(P<0.01)comparedtoKP-Tx

(38.1

±0.1), K-Tx (38.5±0.9), CU (38.0±0.4), and CON (39.5±0.7 ,umol.*m-2^_min-').

During study 1totalleucine fluxdecreased less(P<0.01) inIDDUP(from 28.7±0.8to20.6±0.7)comparedtoKP-Tx, K-Tx,CU,and CON(from38.1 ±0.1to 18.9±0.4,38.5±0.9to 25.1±0.7, 38.0±0.4 to 18.8±0.5, and 39.5±0.7 to 26.6±0.3

Omol.

m-2.

min'-,

respectively). During study 2, IDDUP showed a 56% increase of total leucine flux (28.7±0.8 to 44.9±3.1 jUmol. m-2.min'- ). This increasewassimilartothat in KP-Tx(from42.8±1.7to84.4±2.6), K-Tx(from 37.8±1.1 to 75.7±8.2), CU (37.9±0.5 ^to 78.5±7.9), and CON (41.5±0.6to69.4±3.1

'Rmol.

m2.

min-').

Endogenous leucine

flux

(ELF) (protein catabolism) (Ta- ble IV).Theendogenousleucineflux(which providesamea- sureofprotein

degradation)

isequaltothetotal leucine flux in the basal state as well as in the studies performed without aminoacidinfusion.

Instudy 2, the combinationofhyperinsulinemiaandhy-

peraminoacidemia

causedasimilar absolutedecline of ELF in IDDUP (28.7±0.8 to 4.8±0.5

jtmolm-2-min

'), KP-Tx (42.8±1.7 to 20.5±0.5), K-Tx (37.81.1 to 19.90.9), CU (37.90.5 to 22.31.1), and CON (41.5±0.6 to 18.9±0.9

,mol

*m-2.

min-').

Leucineoxidation (LO)

(Table

IV). Inthe

postabsorptive

state, leucine oxidation was similar in IDDUP

(8.0±0.1 ,umol*.m-2. min-'),

^KP-Tx (9.8+0.3), K-Tx (7.5±0.3), CU (6.6±0.4), and CON (7.5±0.1

,umol.m-2.min-'). During

study 1 both IDDUP(from 8.0±0.1 to 8.5±0.3

,M1mol. m2_

min-')

and K-Tx (from 7.5±0.3 to 6.1±0.4

,umol m2

min'-) demonstratedadefective

suppression

ofleucineoxida- tionwhencomparedtoKP-Tx

(from

9.8±0.3to4.6±0.2),CU (from 6.6±0.4 to 3.1±0.1), and CON (from 7.5±0.1 to

5.1±0.1

4mol

^.m-2.

min'

).During study2the leucineoxida- tion increased less in IDDUP

(from

5.0±0.4 to 17.0±0.5 ,umol*m2

*min-')

with respect to KP-Tx

(6.0±0.8

to

40.1±0.4), K-Tx

(6.9±0.9

to 35.6±1.2), CU (7.0±1.1 to

33.2±1.0), and CON

(7.3±0.2

to 33.3±0.2

zmol

m2.

min-').

Nonoxidative leucine

disposal (protein

synthesis)

(NOLD)

(TableIV).Inthe

postabsorptive

statetheNOLD(anindexof protein synthesis) was significantly reduced in IDDUP (20.7±0.2,mol m-2*min-')with respecttonormalcontrols (32.0±0.7

Mmol.m-2min-';

P < 0.01). KP-Tx

(28.3+0.6 ,umol

^-m-2

min-'),

K-Tx(31.0+1.3 imol. m-2.min-'),and CU(31.4±0.1 Amol*m-2^_

min-')

demonstratednodifference in NOLDwith respecttonormals(P=NS).Duringtheinsulin clamp(study 1),NOLDdecreasedlessinIDDUP

(20.7±0.2

to

12.1±0.4) than in KP-Tx

(28.3±0.6

to 14.3±0.3), K-Tx (31.0±1.3 ^to 19.0±0.9), CU

(31.4±0.1

to 15.7±0.5), and

% _

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2

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+1 +1 +1 +1

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CON (32.0±0.7 to 21.5±0.6). During study 2, NOLD rose significantly above the basal rate in KP-Tx (from 34.1±1.1 to 44.3±0.8), K-Tx (30.9±1.2 to40.1±2.7), CU (30.5±1.9 to 45.3±2.9), and CON (from 31.2±0.8 to 36.1±0.8), while in IDDUP(from 24.1± 1.1 to 27.9±0.7; P=NS)it did not show anysignificant change from the basal value.

Netleucine balance.Inthe basal state all study groups dem- onstrated a similar negative flux of leucine into and out of proteins, averaging -8.0±0.1, -9.8±0.3, -7.5±0.3, -6.6±0.4, and -7.5±0.1 utmol-m-2 -min- in IDDUP, KP-Tx, K-Tx, CU, andCON, respectively. During study 1, the net leucine balance did not change from the basal value in IDDUP (-8.5±0.3 ,umol^-m-2* min-'),whileit became lessnegative inKP-Tx(-4.6±0.2), K-Tx (-6.1±0.4), CU(-3.1±0.1), and CON(-5.1±0.1)compared with the value in the basal state.

During study 2, the combinedhyperaminoacidemia/hyperin- sulinemia induced a similarpositivenetleucine balance in ID- DUP(from-5.0±0.4 to23.1±0.9

Amol

* m-2*min-'),KP-Tx (from-6.0±0.8 to 23.8±1.9), K-Tx (-6.9±0.9 to20.2±2.0), CU (-7.0±1.1 to 23.0±2.1), and CON (from -7.3±0.4 to

17.2±1.6

gmol

*m-2*min-').

Leucine turnoverbasedonKIC specificactivity(TablesIV andV). Because it has been suggested that plasma KIC specific activity may provide a more accurate index of the intracellular precursor pool involved inleucine turnover, all calculations were repeated utilizing the steady-state plasma KIC specific activity achieved during the last hour of the basal and experi- mentalperiods. Table IV shows that the qualitative changes in leucine turnover (LO, ELF, and NOLD) calculated with the plasma KICspecific activity (reciprocal pool model), closely parallel thosedeterminedwith the plasma leucinespecific activ- ity(primary pool model) under the present experimental con- ditions. During combined hyperinsulinemia/hyperaminoaci- demia the NOLD wasdefectivelystimulated in IDDUP(from 30.5±2.2 to 28.6±0.5

gmol

*m-2.

min-'),

while it showed a

similarstimulationinKP-Tx,K-Tx,

CU,

andCON

(49.4±2.1,

54.2±5.4,60.7±5.1 and54.4±1.6,respectively).

Glucosemetabolism. Duringthe120-180 min of

study

1 in IDDUP, theglucose infusionrate

required

tomaintain

eugly-

cemia was 3.4±1.6

mg-kg-'-min-1, significantly

less

(P

<0.01 )thanthe other groups.AfterKP-Txandafter

K-Tx,

the glucose infusion rate required to maintain

euglycemia

in- creasedto6.0±0.8and4.2±0.9

mg. kg-'

*

min-', respectively.

CU patients' insulin-stimulated glucose metabolism^was still defectivewith respecttoCON

(normals) (6.2±0.2

vs. 7.5±0.3 mg *

kg-'

^-

min'

).

During study 2 the glucoseinfusion ratesinIDDUP,KP- Tx, K-Tx, CU, and CON were 3.2±1.2, 6.3±1.5, 5.9±0.9, 6.5±0.6, and 7.1±0.4 mg

kg1'-minm', respectively.

These rates were similartothose observedwhen theinsulin clamp studywasperformedwithoutamino acid infusion

(P

⁼NSvs.

study 1 forallgroups).

Discussion

Theoverallincidenceof diabeticnephropathyin thediabetic populationis 35% in Westerncountries(31 ).All

patients

who developproteinuriaenter a

hemodialysis

programorgetakid- neytransplant within 5-10years(32). Isolatedkidneytrans- plantationisstillthefirst choice

surgical

approach fortype 1 uremic-diabetic patients (33). One ofthe major drawbacks ofisolatedkidneytransplantis the needtocontinuelife-long therapy with prednisone (10

mg.d-'), cyclosporine

(5 mg*

kg-'

*

d'

), andazathioprine( 1 mg * kg- *

d'-l)

ininsulin- dependentdiabetic patients ( 13, 34). Duringthe lasttwode- cades theimprovementof bothpatientand organsurvival has made it possible to extend pancreas transplant, alone or in combinationwith thekidneyto agreaternumberofpatients (34). Infact,bothsegmental andtotal pancreastransplanta- tion are capable of maintaining long-term insulin indepen-

TableV.Metabolic andKinetic ParametersofK-TxandCUduringCombinedHyperinsulinemia-Hyperaminoacidemia

K-Tx CU

Bas Stu Bas Stu

Patients (n) 4 4

Hbu, (%) 7.8±0.5* 5.8±0.4

Plasma glucose(mg/dl) 120.7±10.8* 95.5±3.6 84.7±3.6 88.3±3.7

Plasmaleucine

(&M)

^104±8 207±23* 110±9 209±17$

Amolbm-2 min-'

ELFICU 37.8±1.1

19.9±0.9*

37.5±0.5

22.3±1.1*

ELFkj, 54.0±2.6

39.3±5.4*

56.8±2.1 45.9±5.2*

LOI.U 6.9±0.9

35.6±1.2*

7.0±1.1

33.2±1.0*

Lold. 9.9±1.4

50.9±3.6*

10.6±2.2

51.4±3.8*

NOLDkuJ

30.9±1.2 40.1±2.7* 30.5±1.9 45.3±2.9*

NOLDEc 44.1±2.9 54.2±5.4* 46.2±2.7 60.7±5.1*

NLBI.J

^{-6.9±0.9 20.2±2.0* -7.0±1.1} 23.0±2.0*

NLB~C -9.9±1.4 14.9±0.8* -10.6± 1.9 14.8±1.1*

Dataareexpressed using both the leu and a-KIC specific activities.

*P <0.01 compared to CU. *P<0.01 compared to basal.

1954 Luzietal.

dence (1-10 years) ( 13, 35).Inaddition, previousstudies have demonstrated that transplantation ofthepancreas along with thekidneyhas a betterimpacton the quality oflife of insulin- dependentdiabetic patientscompared withtransplantation of thekidneyalone(36, 37).

In the present study we used labeled leucine to assess basal andinsulin-stimulatedwhole body proteinmetabolism in ID- DUP beforeandafter isolatedkidney transplant or combined pancreasandkidney transplant. Ourdatademonstrate that the combinationof diabetes mellitusanduremiacauses a serious impairment ofthebasal andinsulin-stimulatedprotein metabo- lism,thatkidneytransplant only partially reverts the

protein/

metabolic alterationsofIDDUP, andfinally,thatthe combina- tion of the replacement of the kidneyand the pancreas leads to anearnormalization of whole-body protein metabolism, de- spite the needofchronicimmunosuppressivetherapy.

Thenitrogen excretionrate wassignificantlyhigher in the basalstatein IDDUP comparedtothe othergroups.This indi- catedanegative nitrogen balance before transplantationwhich was reverted by combined kidney and pancreas transplanta- tion (TableI). Theproteincontentofthedietwas comparable in IDDUP, inK-Txalone, and inKP-Tx.Therefore,thediffer- enceinnitrogenexcretionrate was notattributableto adiffer- ence in diet. Transplanted kidney may present a relapse of diabetic nephropathy. In such cases, in ordertoprevent the progression ofrenal damagealow-protein diet should be ad- ministered (38). A recent reportby Brodsky et al. (39) has shown that a lowprotein dietmay cause an impairment of leucine metabolism andaworsening of nitrogen balance and nutritionalparameters.Therefore,apossible advantageofper- formingthetransplant of thepancreasalongwith thekidneyis thatit isnotnecessarytogiveaprotein restricteddiettoIDDM patients after the transplant andduringchronicprednisonead- ministration.

In apreviousreport weshowed thatIDDMinpoor meta- bolic control is characterizedbyincreased basal ELF (proteoly- sis), LO, and NOLD (an index ofprotein synthesis)(4). The amelioration of the metabolic control byoptimizingconven- tional insulintherapy normalized basal ELF,NOLD,andLO inIDDMsubjects (4). The basicdefectinprotein metabolism in type 1 diabetes mellitus would thereforeseemto beapri- maryincrease inproteolysis.Inaddition,inIDDM, the stimula- tion ofprotein synthesisby combinedhyperinsulinemia/hy- peraminoacidemiais similartothat in normals.In contrast,we havepreviouslyshownthatpatientswithchronicuremiaare characterized by reduced amino acid concentrations andre- duced proteolysis (ELF), LO, and NOLD (8). In the same study, theprimarydefect in chronic uremiawasfoundtobeat thelevelofproteinsynthesis,while insulin actiononproteoly- siswasfoundtobenormal. Previous reportshad shown that hemodialytictherapy(40)and chronicambulatoryperitoneal dialysis(41)are notcapable ofnormalizing proteinturnover andinsulin actioninuremicpatients.Possibleexplanationsof theparticularlylow ELFandNOLD inpostabsorptiveIDDUP when compared to controls include the following: (a) in- creasedinsulinlevelswithrespecttonormals(8),

(b)

increased growthhormoneconcentration(Table II),and(c)increased epinephrineconcentration. In

particular

both

high

insulin(4, 6)andgrowthhormone(42)concentrationshave beenlinked tothe lowleucine fluxes.We have

previously

shown that

epi-

nephrine

infusionin normals^{causes a}mild decline of amino acidconcentrations

(43).

^{From the}present results itseemsthat IDDUP

patients

demonstrate intracellular defects localized bothatthe

proteolytic

and

proteosynthetic pathways.

Thisex-

plains

the

striking negative nitrogen

balance in such

patients.

Apparently

both the

anti-proteolytic

andthe

proteosynthetic

defectsare

acquired.

Insulin

deficiency

is the

primary

causeof

increaseof

proteolysis

in

fasting

IDDM

(4);

whenIDDM pa- tients

develop

uremiaa newdefect in

protein synthesis

may be

selectively

determined.

During

exogenous insulin infusion

(study 1)

the inhibition ofELFisdefective in IDDUP^com-

pared

tothe othergroups

indicating

aninsulin resistance with respecttothe

proteolytic pathway, only

whendiabetes mellitus anduremiaarecombined.

During study

2 thestimulation of

protein synthesis

wasdefective in IDDUP

compared

toall the othergroups.

Therefore,

the

higher nitrogen

excretionratepres- entin IDDUP is due

partially

toadefective inhibition ofpro-

teolysis

and

partially

toadefective stimulationof the

protein synthetic pathway by

insulin. Thecombinationof both defects makesmoreaminoacid

nitrogen

availablefor the intracellular oxidativeprocesses.

*Patients

after successful

kidney transplant

havetocontinue conventional insulin

therapy

with theadditional

problem

of chronic

immunosuppressive drugs. Furthermore,

Nair et al.

(44)

have

recently

shownthat

hyperglucagonemia

incombina- tion with insulin

deficiency (the

hormone-metabolic condition indiabetic

patients

afterisolated

kidney transplant)

accelerates

protein

catabolism. Insuch

patients (K-Tx)

^{the basal}ELFis similarorincreased with respectto normals

depending

upon the

degree

ofinsulinization. Patientswithsuccessfulpancreas and

kidney transplant

areinsulin

independent

andare

capable

of

overcoming

thealterations determined

by

thesteroidther- apyon

glucose

metabolism

by

^meansof basal andpost-pran- dial

hyperinsulinemia (9, 10, 12, 35,45). During study

1

(insu-

lin

infusion)

thedefect of

endogenous

leucine flux

(proteoly- sis)

of IDDUP is reverted inK-Tx. Incontrast,

insulin-induced

inhibitionoftheleucine oxidative

pathway

isstill defective in K-Tx.

Therefore,

isolated

kidney transplant

mayrevert most

of the intracellular metabolic

pathways,

with the notable ex-

ception

ofinsulin-stimulated

suppression

of LO. This

finding

may be due to a direct

glucocorticoid

activation of muscle branched-chain a-ketoacid

dehydrogenase (46).

Thefact that K-Tx havean absolute value of NOLD similartoCON and

higher

than KP-Tx

during hyperinsulinemia

maybe

explained

asfollows:

(a) patients

with isolated

kidney transplant

^aregen-

erally

insulin resistantas

regards

whole

body

leucine metabo-

lism, although

the

impaired

insulin action reaches statistical

significance only

for

LO;

incontrast,

patients

with combined pancreas and

kidney transplantation apparently

havean in- creased insulin

sensitivity

^{in the}

protein

metabolic

pathways;

(b) considering

thevariation from basal instead ofthe absolute value of

NOLD, only

inIDDUPthe decreaseis

significantly

lower than

CON; (c)

in orderto correctfor the fall in amino

acids,

thebestassessment of

protein synthetic capacity

isob- tained ^{in a} condition of combined

hyperinsulinemia/hyper-

aminoacidemia

(4).

Inour 14

patients

after combined

kidney-pancreas

trans-

plantation,

we foundaclear-cutdissociationbetween insulin effectson

glucose

and

protein

metabolism. In

fact,

aftercom-

bined

transplantation glucose

metabolism is

impaired,

while