20. Lindholm A, Kahan BD. Influence of cyclosporine pharmacoki-netics, trough concentrations, and AUC monitoring on outcomeafter kidney transplantation. Clin Pharmacol Ther 1993; 54:205.

21. Lindholm A, Ohlman S, Albrechtsen D, et al. The impact of acuterejection episodes on long-term graft function and outcome in1347 primary renal transplants treated by 3 cyclosporine reg-imens. Transplantation 1993; 56: 307.

22. Kahan BD, Kramer WG, Wideman C, Flechner SM, Lorber MI,Van Buren CT. Demographic factors affecting the pharmaco-kinetics of cyclosporine estimated by radioimmunoassay.Transplantation 1986; 41: 459.

23. Zimmerman J, Kahan BD. Pharmacokinetics of sirolimus instable renal transplant patients after multiple oral dose ad-ministration. J Clin Pharmacol 1997; 37: 405.

24. Dantal J, Hourmant M, Cantarovich D, et al. Effect of long-termimmunosuppression in kidney-graft recipients on cancer inci-dence: randomised comparison of two cyclosporin regimens.Lancet 1998; 1: 623.

25. Ponticelli C, Minetti L, Di Palo FQ, et al. The Milan clinical trialwith cyclosporine in cadaveric renal transplantation. A three-year follow-up. Transplantation 1988; 45: 908.

26. The Canadian Multicentre Transplant Study Group. A random-ized clinical trial of cyclosporine in cadaveric renal transplan-tation. Analysis at three years. N Engl J Med 1986; 314: 1219.

27. Weidle PJ, Vlasses PH. Systemic hypertension associated withcyclosporine: a review. Drug Intell Clin Pharm 1988; 22: 443.

28. Vathsala A, Weinberg RB, Schoenberg L, et al. Lipid abnormal-ities in cyclosporine-prednisone-treated renal transplant recip-ients. Transplantation 1989; 48: 37.

29. Kasiske BL, Tortorice KL, Heim-Duthoy KL, Awni WM, Rao KV.The adverse impact of cyclosporine on serum lipids in renaltransplant recipients. Am J Kidney Dis 1991; 17: 700.

30. MacDonald AS for the Rapamune Global Study Group. A ran-domized, placebo-controlled trial of Rapamune in primary re-nal allograft recipients. Abstracts of the Transplantation So-ciety XXVII World Congress, Montreal 1998 (Abstract 426).

31. Kahan BD for the Rapamune U.S. Study Group. A phase IIIcomparative efficacy trial of Rapamune in renal allograft re-cipients. XVII World Congress, Montreal, Canada 1998 (Ab-stract 198).

Received 4 March 1999.Accepted 11 May 1999.

0041-1337/99/6810-1532/0TRANSPLANTATION Vol. 68, 1532–1541, No. 10, November 27, 1999Copyright © 1999 by Lippincott Williams & Wilkins, Inc. Printed in U.S.A.

THE EFFECTS OF MAINTENANCE DOSES OF FK506 VERSUSCYCLOSPORIN A ON GLUCOSE AND LIPID METABOLISM AFTER

ORTHOTOPIC LIVER TRANSPLANTATION1

LUIS A. FERNANDEZ,2–4 ROGER LEHMANN,2 LIVIO LUZI,5 ALBERTO BATTEZZATI,5 MARIA C. ANGELICO,2

CAMILLO RICORDI,2–4 ANDREAS TZAKIS,4 AND RODOLFO ALEJANDRO2,3,6

Diabetes Research Institute University of Miami School of Medicine, Veterans Administration Medical Center,Department of Surgery, University of Miami School of Medicine, Miami, FL and the Department of Medicine,

Istituto Scientifico, San Raffaele Milano, Italy

Background. Posttransplant diabetes mellitus(PTDM) has gained widespread attention due to themicro and macro-vascular complications that increasethe morbidity and mortality of patients receiving solidorgans. The higher incidence of PTDM has beenmainly attributed to the immunosuppressive therapy.Therefore, this study compares the metabolic side ef-fects of low dose maintenance therapy of FK-506 andCyclosporin A (CsA) in 14 patients 1 year after ortho-

topic liver transplant and analyzes possible factorsthat contribute to the development of PTDM.

Methods. Two groups (n57) differing in their immu-nosuppressive regimen (FK506 or CsA) were matchedto eight control subjects and compared to each other.The effects of in vivo insulin action were assessed bymeans of the euglycemic hyperinsulinemic clamptechnique. Arginine stimulation tests at normo- (5.5mM) and hyperglycemic (15 mM) levels were per-formed and the acute insulin, C-peptide, and glucagonresponse (2–5 min) to arginine were determined.

Results. Insulin sensitivity (total glucose disposal)was statistically lower in patients treated with FK-506and CsA (5.0560.47 and 5.0560.42 mg/kg/min) as com-pared to controls (6.6260.38 mg/kg/min) (P<0.02), witha significantly higher nonoxidative glucose disposalfor the control group (P<0.01), and lower free fattyacid levels (P<0.05). Absolute values for acute insulinresponse were higher but not significantly differentfor the transplanted groups. The lower percentage ofincrease of insulin release after arginine stimulationobserved in the FK-506 and CsA groups as compared

1 This work was supported in part by NIH grant DK25802,MO1RR0528, The Diabetes Research Foundation, and a scholarshipfrom the Swiss National Science Foundation

2 Diabetes Research Institute University of Miami School of Med-icine.

3 Veterans Administration Medical Center.4 Department of Surgery, University of Miami School of Medicine,

Miami, FL.5 Department of Medicine, Istituto Scientifico, San Raffaele Mi-

lano, Italy6 Address correspondence to: Rodolfo Alejandro, MD, Diabetes

Research Institute (R-14), 1450 NW 10th Avenue, Miami, Fl 33136.

TRANSPLANTATION1532 Vol. 68, No. 10

with controls (754%6100, 644%6102 vs. 1191%6174)(P<0.03 and 0.02, respectively), suggests a reduced bcell secretory reserve in both treated groups. Also, theacute glucagon response to arginine during hypergly-cemia declined less in the FK-506 (28%) and CsAgroups (29%) compared with controls (48%) (P<0.05)indicating a defect in the pancreatic b cell-a cell axis.

Conclusions. There are no major metabolic differ-ences on low maintenance doses between FK-506 andCsA. Both immunosuppressant agents contribute tothe development of PTDM at different levels.

With increasing survival and better long-term outcomes inpatients after orthotopic liver transplantation (OLTx*), theinterest in common metabolic abnormalities and the sideeffects associated with the commonly used immunosuppres-sive drugs such as diabetes, hypertension, dyslipidemia, andobesity has increased substantially (1). In particular, post-transplant diabetes mellitus (PTDM) has gained widespreadattention due to the micro- and macro-vascular complicationsthat increase the morbidity and mortality of patients receiv-ing solid organs. Diabetes mellitus has been reported to beassociated with an increased incidence of rejection episodes,a higher mortality rate during the first years after livertransplantation (2), an increased risk for coronary heart dis-ease (3), and a higher incidence of bacterial and fungal infec-tions (4). The higher incidence of PTDM has been mainlyattributed to the immunosuppressive therapy. Several stud-ies have demonstrated that corticosteroids (5, 6), cyclosporinA (CsA) (7–9) and FK-506 (tacrolimus) (9–12) are among thedrugs that significantly contribute to a disturbed glucosehomeostasis. The reported incidence of PTDM when usingCsA or FK506 as immunosuppressive agents varies between30 to 40%. Forty percent of patients with PTDM requireinsulin therapy (7, 13). The pathogenesis of diabetes in CsA-or FK506-treated patients has been attributed to reductionin the DNA synthesis and mRNA content of pancreatic islets(14), a reduction in the islet capacity to release insulin (15,16), and a reduction in insulin synthesis (14). A possible roleof calmodulin in insulin secretion has also been suggested(17, 18). Although CsA and FK-506 have similar mechanismof action (14, 18, 19), some studies reported glucose metabolicdisorders to be more common for patients receiving tacroli-mus versus CsA (13, 20), although in other studies the inci-dence of hyperglycemia and diabetes has been similar withboth drugs (11, 21). Data from small and large animal exper-iments suggest that FK506 causes a dose-dependent toxicityto b cells (16, 22). Impairment in insulin secretion and insu-lin content of the b cells in animals treated with FK506correlate well with morphological changes in b cells, and isreversible 14 days after withdrawal of the drug (23). Severalpatients were described in the literature where PTDM in-duced by FK-506 could be totally reversed when patientswere switched to CsA (13). Although some reports showedthat FK-506 and CsA induced a similar glucose intoleranceand hyperinsulinemia after successful allogeneic liver trans-plantion (9, 11, 20, 21, 24), other studies showed a higherincidence of hyperglycemia or diabetes in FK506-treated pa-tients (9, 13). In contrast, two other studies in liver trans-

plant recipients did not show any significant diabetogenicside effect (12, 25) or dose dependency of FK506 treatment(26).

In view of these contradictory results and to assess thelong-term metabolic consequences, we evaluated insulin sen-sitivity, a and b cell function, and b cell reserve in livertransplant recipients in clinical stable condition with no orminimal doses of steroids (five patients were off steroids inthe FK506 group and three patients in the cyclosporinegroup) on low-dose maintenance doses of FK-506 or CsA asprimary immunosuppressive agents.

MATERIALS AND METHODS

Subjects. A total of 14 patients in stable clinical condition wasstudied after orthotopic liver transplantation. All patients weretransplanted for a histologically confirmed liver cirrhosis due tohepatitis C. Stable clinical condition was defined as normal liverfunction tests, no rejection episode for at least 6 months, no recyclingof steroid doses after rejection, and no current opportunistic infec-tions. Patients with diabetes mellitus according to the new diagnos-tic criteria for diabetes (including impaired fasting glucose) wereexcluded (27). Seven patients received CsA at a mean daily dose of3.366(SEM) 0.57 mg/kg/day and seven received FK-506 at a meanmaintenance dose of 0.0560.01 mg/kg/day. Patients were matchedwith regards to prednisolone doses (1.360.3 vs. 1.160.15 mg/day),age (49.365.2 vs. 47.063.3 years), body mass index (26.160.5 kg/min(2) vs. 25.760.5 kg/min (2), lean body mass (LBM) in kg (56.764.2 vs.55.865.8 kg), gender (five males/two females) and time after trans-plant (324627 vs. 34068 days). Controls (hepatitis C negative) werematched regarding body mass index (25.460.3 kg/m (2), LBM(58.862.9 kg), and age (48.164.0 years) and gender (six males/twofemales) as shown in Tables 1 and 2.

The nature, purpose, and risk of the studies were explained to allsubjects before the study and their informed written consent wasobtained. The study was approved by the Ethical Committee at theUniversity of Miami.

Subjects were admitted to the ward the morning of the study.Patients were maintained on a diet of at least 250 g of carbohydratesand 70–80 g of proteins per day for 2 weeks preceeding the study. Allstudies were performed after a 10- to 12-hr overnight fast. Before thestudy, two plastic angio-catheters were placed into forearm veins,one of them placed in an antecubital vein for the infusion of glucose,insulin, and arginine and a second catheter was inserted retro-gradely into a wrist vein for blood sampling. This forearm was placedin a warming chamber heated to 60°C at least 30 min before bloodsampling, to allow arterialization of the venous blood. After a prim-ing dose of 5 mg/kg of body weight, a prime-continuous infusion of[6,6-d2]-glucose (Masstrace Inc., Woborn, MA) was administered(0.045 mg/kg/min) for the next 270 min. After a tracer equilibrationperiod adequate to achieve tracer’s steady state in plasma (120 min),all study subjects underwent a euglycemic hyperinsulinemic clampas previously described (28, 29) (Fig. 1). In brief, after a priming doseof 80 mU insulinzsqm21zmin21 for 10 min a continuous infusion ofregular human insulin (Eli Lilly, Indianapolis, IN) was administeredat a rate of (40 mU insulinzsqm21zmin21) for 140 min to achieve andmaintain an increase in plasma insulin concentration between ;75to 100 mU/ml. To maintain plasma glucose constant at 5.5 mM, a 20%dextrose solution (McGaw Inc. Irvine, CA) was infused. Adjustmentsin the rate of infusion were based on a negative feed-back principle,depending on the blood glucose determinations every 5 min. Plasmaglucose was measured at bedside using a glucose analyzer (Model2300 Stat, Yellow Springs Instrument Co., Inc, OH). Plasma glucoseand plasma deuterated glucose enrichments were assessed every 10min in the last hour of the basal equilibration period and during the150 min of the euglycemic clamp study. During the euglycemicclamp, the plasma-free insulin was measured every 10 min, and

* Abbreviations: OLTx, orthotopic liver transplantation; AGR,acute glucagon responses BW, body weight; CsA, cyclosporin A;LBM, lean body mass; PTDM, posttransplant diabetes mellitus.

FERNANDEZ ET AL.November 27, 1999 1533

measurements of free fatty acid, C-peptide, glucagon, and glycerol at30-min intervals through-out the clamp study.

Indirect calorimetry. During all clamp studies continuous indirectcalorimetry was performed and carbon dioxide production and oxy-gen consumption were measured as described elsewhere (30, 31) toestimate net rates of carbohydrate and lipid oxidation as previouslydescribed (32). Briefly, a transparent air-tight plastic ventilatedhood was placed over the head of the subject, keeping a slightnegative pressure to prevent the loss of expired air. Minute byminute measurements of carbon dioxide consumed and the oxygencontent of the expired air were analyzed by model Vmax 29 (SensorMedics, Yorba Linda, CA) during the baseline period (30 min) andthe next 150 min of euglycemic-hyperinsulinemic clamp.

Body composition. Body composition was assessed by multi-fre-quency bio-impedance (Bodystat MultiScan 5000, Bodystat LTD, Isleof Man,UK). Regression equations are derived which relate imped-ance to fat free mass or total body water measured by independenttechniques.

Arginine-stimulated insulin secretion at normo- and hyperglyce-mic levels. Five grams of L-arginine hydrochloride 10% were giveni.v. over a 30-sec period (Rgene, Pharmacia Inc, Clayton, NC) (33).

Arterialized blood samples for analysis of glucose, insulin, glucagon,and C-peptide were obtained at 210, 25, 0, 2, 3, 4, 5, 7, 10, 15, 20, 25,and 30 min for the evaluation of a and b cell functions. To increasethe sensitivity to detect subtle defects in the b cell reserve and toevaluate the effects of glucose potentiation on arginine-induced in-sulin secretion, glucose was clamped for 45 min at 15 mM plasmaglucose. The priming dose was calculated according to a modifiedalgorithm (34) using an infusion of 20% dextrose solution for 15 min.Then, glucose infusion was monitored every 5 min and the glucoseinfusion rate was adjusted to maintain the hyperglycemic level at 15mM. After 45 min of hyperglycemic clamp, prestimulated blood sam-ples were obtained at 210, 25, and 0 min, and a second 5-g arginineinjection was given. Samples are obtained at the same time intervalsas described above.

Analytical determinations and calculations. Blood samples werecentrifuged immediately at 4°C and stored at 220°C until analysis.Free insulin was measured by means of radioimmunoassay doubleantibody (Linco Research, Inc, St Charles, MO). Glucagon and C-peptide were measured using a RIA Kit (Diagnostic Products Corpo-ration, Los Angeles, CA). Deuterated glucose enrichments were mea-sured by means of a gas-chromatography/mass spectrometry using a

TABLE 1. Baseline clinical and biochemical parameters of liver transplant recipients on tacrolimus (FK-506), cyclosporin A (CsA), andnormal subjects

Groups FK-506 CsA Control

Age (yr) 47.063.3 49.365.2 48.164.0Sex (male/female) 5/2 5/2 6/2Immunosupressant dose (mg/kg/day) 0.0560.01 3.3660.57 N/AMethyl-prednisolone (mg/day) 1.160.75 1.360.60 N/AFollow-up post-transplant (days) 34068 324627 N/ABMI (kg/m2) 25.760.5 26.160.5 25.460.3LBM (kg) 56.764.2 55.965.8 56.862.9Mean arterial blood pressure (mm Hg) 98.164.55 99.863.86 97.863.9FBG (mM) 6.160.2 5.560.1 5.760.1Hb A1C (%) n54.0–6.0% 5.260.2 5.360.3 5.860.1Serum creatinine (mmol/L) 141614 164610 12569Creatinine clearance (1.73 m2) 52.465.8a 56.362.6b 103.967.1ASP (SGOT) U/L 49613 8769 31610Direct bilirubin mg/dl 0.7060.09 0.6860.10 0.4860.11Alkaline phosphatase U/l 100614 154623 6369Nitrogen collection (g/24h) 9.0760.75 10.7361.20 15.0061.43

Values are means 6SEM.a FK-506 versus control P,0.01.b CsA versus control P,0.01.

TABLE 2. Comparison of metabolic parameters assessed during euglycemic hyperinsulinemic clamp and indirect calorimetry betweengroups

Parameters FK-506 CsA ControlP

(FK-506 vs.control)

P(CsA vs.control)

Body weight (BW; kg) 76.7613.0 78.2616.9 75.763.7 NS NSLean body mass (LBM; kg) 56.7611.4 55.9615.4 58.567.9 NS NSGlucose infusion rate (mg/min) 4476171 4526151 472696 NS NSTotal glucose disposal (mgzkg BW21zmin21) 5.0560.47 5.0560.42 6.6260.38 ,0.03 ,0.03Baseline (230–0 min) hepatic glucose

production (HGP) (mgzkg BW21zmin21)1.9760.08 1.8560.12 2.0260.13 NS NS

HGP from 90–150 min during euglycemichyperinsulinemic clamp (mgzkg BW21zmin21)

0.0260.02 0.0160.01 0.4360.24 NS NS

Glucose oxidation (mgzkg LBM21zmin21) 2.3560.20 1.9260.55 1.1560.25 ,0.01 NSNon-oxidative glucose oxidation (mgzkg

LBM21zmin21)4.4960.58 5.2160.60 7.3467.34 ,0.01 0.04

Protein oxidation (mgzkg LBM21zmin21) 0.0460.01 0.0560.01 0.0760.02 NS NSBasal energy expenditure (kcal/day) 16326328 13616646 15656134 NS NSBaseline (230–0 min) free fatty acids (mmol/L) 0.5860.23 0.6060.19 0.4260.19 NS NSFree fatty acids (90–150 min) (mmol/L) 0.1960.17 0.0860.05 0.0560.02 0.03 0.06

TRANSPLANTATION1534 Vol. 68, No. 10

technique that is a derivation of the sample by means of boro-acetylation (35, 36). Rates of glucose appearance and disposal werecalculated from the deuterated glucose data in the last 30 min of thebasal period and during the last 60 min of the clamp studies. Duringinsulin infusion, a non-steady-state exists, and therefore calculationswere performed according to Steele’s equation (37). Hepatic glucoseproduction and peripheral glucose disposal were assessed as previ-ously described (28). Urinary glucose loss was negligible in ourpatients. Twenty-four hour urine was collected in all patients andtotal nitrogen in the urine was calculated, assuming a protein oxi-dation equal to protein catabolism and correcting for insensitivelosses as previously described (28, 38, 39).

The rate of glucose and lipid oxidation were calculated in the basalstate and during euglycemic clamp from the nonprotein respiratoryquotation (38, 40) assuming the protein oxidation to be constantduring the duration of the clamp. Nonoxidative glucose disposal wascalculated subtracting the glucose oxidation rate from the total tis-sue glucose disposal. Calculation of glucose, lipid, and protein oxida-tion were expressed per kg of body weight (BW) and of LBM.

Fasting levels of plasma glucose, insulin, and C-peptide werecalculated as the mean of the 215 to 0 min values for the normo- andhyperglycemic arginine stimulation test. The acute insulin, acuteC-peptide, and acute glucagon (AGR) responses to arginine werecalculated as the incremental area under the respective curves be-tween 2–5 min after injection of arginine by means of the trapezoidalrule (41–43). The acute insulin response during glucose potentiationto arginine-induced insulin secretion was expressed as a percentageof basal and calculated as the acute response (mean of the valuesfrom 2–5 min minus the basal value, divided by the basal value, andmultiplied by 100). For the calculation of the acute hormonal re-sponse after arginine stimulation, all patients were included in theirrespective groups, with the exception of three patients from thecontrol group (n58) for which data were not available.

Statistical analysis. Statistical analysis was performed using Sta-tistica for Windows (Version 5.0, StatSoft, Tulsa, OK). All data arepresented as mean6SEM analysis of variance with one betweengroups factor and one repeated measures factor was used. Compar-ison between groups were performed by the Scheffe test. When only

two groups were compared the Student’s t test was used for para-metric and the Wilcoxon sign rank test for nonparametric variables,

RESULTS

Baseline clinical and laboratory parameters of the patientsand controls are summarized in Table 1. The mean fastingplasma glucose levels in the CsA group (5.560.1 mM) wassimilar to values obtained in the FK506 (6.160.2 mM) andcontrols (5.760.1 mM). Values for HbA1c were 5.360.3% forthe CsA group, 5.260.2% for the FK506, and 5.860.1% forthe control group. No statistical significant differences couldbe observed within the three groups with regards to clinicalcharacteristics with the exception of the creatinine clearanceand 24-hr nitrogen collection. Creatinine clearances werefound to be significantly different in the FK-506 and CsAgroup (52.465.8 and 56.362.6 ml/min) compared with thecontrol group (103.967.1 ml/min) (P,0.01). Also the 24-hrnitrogen collection was different in the FK-506 group andCsA as compared with the control group (9.0760.75 and10.7361.20 g/L) vs. 15.061.43 (P,0.002 and 0.07, respective-ly).

Insulin sensitivity. The fasting plasma free insulin con-centration was not statistically different between the threegroups FK-506, CsA, and control (32.564.9, 23.264.5,24.267.1 pmol/L, respectively). Accordingly, basal C-peptidewas not statistically significant for the three groups (412696,321681, and 275695 pmol/L) respectively. During euglyce-mic hyperinsulinemic clamp, the insulin concentration atequilibration was not statistically different among the groups(5096151, 4746165, and 5176270 pmol/L for CsA, FK-506,and control groups, respectively). During the euglycemic-hyperinsulinemic clamp, the concentration of C-peptide inthe CsA group (5246267 pmol/L) and FK506 (5096263.6pmol/L) were not statistically different from the controlgroup (3796185 pmol/L). The plasma glucagon concentrationin the last hour of the clamp was similarly inhibited in theFK506 (22.565.7 pmol/L), in the CsA (23.566 pmol/L) and inthe control group (15.5563.2 pmol/L) (Fig. 2).

Hepatic glucose production and peripheral glucose dis-posal, in the basal state and during insulin infusion. Post-absorptive hepatic glucose output was very similar in pa-tients with liver transplantation treated with CsA (1.856.12mgzkg BW21zmin21), FK-506 (1.9760.08 mgzkg BW21zmin21) compared with control patients (2.0260.13 mgzkgBW21zmin21) (NS). During the last hour of the euglycemic-hyperinsulinemic clamp, the hepatic glucose productionshowed a more pronounced but not statistically differentdecrement in the CsA (0.0160.01 mgzkg BW21zmin21) andFK506 group (0.0260.02 mgzkg BW21zmin21), as comparedto the controls (0.4360.24 mgzkg BW21zmin21).

The total amount of tissue glucose disposal in the last hourof the euglycemic-hyperinsulinemic clamp was calculatedand expressed as per kg of BW. There is an impairment in thetotal amount of total glucose disposal in both the liver trans-plant recipient group CsA (5.0560.42 mgzkg BW21zmin21)and FK-506 (5.0560.47 mgzkg BW21zmin21) as compared tothe control group (6.6260.38 mgzkg BW21zmin21) (P,0.03).Another indicator of insulin resistance is the calculation ofnonoxidative glucose metabolism expressed in relation to thelean body mass (LBM). There is a significant decrease in theamount of nonoxidative glucose metabolism in the FK-506(4.4960.58 mgzkg LBM21zmin21) and CsA (5.2160.60 mgz kg

FIGURE 1. Experimental protocol for assessment of insulin sensitiv-ity and insulin secretory capacity. The euglycemic-hyperinsulinemicclamp was performed using an insulin infusion at a rate of (40mU/m2/min), with a simultaneous infusion of a 20% glucose solutionto keep the plasma glucose concentration at the basal level. Deuter-ated glucose was infused to assess hepatic glucose production andindirect calorimetry was performed throughout the study. A wash-out period followed the 150-min euglycemic-hyperinsulinemic clamp.Insulin secretory capacity of the beta cell was then evaluated byarginine (5 g) injection i.v. at normoglycemia (5.5 mM). Glucose wasthen clamped at 15 mM and after 45 min another arginine stimula-tion test was performed.

FERNANDEZ ET AL.November 27, 1999 1535

LBM21zmin21) as compared to the control group (7.3460.65mgzkg LBM21zmin21) (P,0.01 and 0.04, respectively). Nodifferences were observed when both transplanted groupswere compared.

Free fatty acids concentration in the basal state and duringeuglycemic clamp study. Basal free fatty acids concentrationwere similar in patients treated with FK-506 (0.5860.23mmol/L) when compared with patients treated with CsA(0.6060.19 mmol/L) and control patients (0.4260.19 mmol/L).During the euglycemic hyperinsulinemic clamp, the inhibi-tion of free fatty acid concentration for the last hour of theeuglycemic-hyperinsulinemic clamp was not statistically sig-nificantly different when FK-506 and CsA were compared(0.1960.17 vs. 0.0860.05 mmol/L, respectively). However,when both transplanted groups were compared to the con-trols (0.0560.02 mmol/L) a statistical significant differencewas observed when compared to FK-506 (P,0.03) and almostapproached significance when compared with CsA (P,0.06).

Protein oxidation. When protein oxidation was comparedbetween the three groups, no statistical significant differ-ences were observed when the FK-506 group was compared

to CsA group (0.0460.01 vs. 0.0560.01 mgzkg LBM21zmin21). When FK-506 was compared with the control group(0.0760.02 mgzkg LBM21zmin21), however, there was a sta-tistical significant difference (P,0.01).

b cell function. Fasting plasma glucose levels and meanfasting insulin levels before arginine stimulation test werenot significantly different between the three groups.

The absolute levels and the levels expressed as the per-centage of basal insulin after stimulation with arginine at 5.5and 15 mM glucose are summarized in Table 3. Levels ofinsulin, C-peptide, and glucagon for the three groups arepresented. During the normoglycemic phase of the clampstudy the baseline insulin values did not show any significantdifferences between the three groups (Table 3A). After stim-ulation with arginine, the percentage of basal insulin se-creted was significantly higher in both transplanted groupsas compared to controls. In healthy control subjects, there isa strong correlation between fasting insulin level and acuteinsulin response to arginine at normoglycemia (r50.96,P,0.001). These correlations were also significant in livertransplant recipients treated with CsA (r50.90, P,0.03), but

FIGURE 2. Plasma glucose (A), C-peptide (B), insulin (C), and glucagon (D) profiles during fasting euglycemic-hyperinsulinemic clamp. Thesteady state postabsorptive concentration as well as the time-dependent variation of the hormones are represented for the three differentgroups.

TRANSPLANTATION1536 Vol. 68, No. 10

not significant for FK506 (r50.21, P,0.6) (Fig. 3). At hyper-glycemic levels this correlation was even stronger (r50.89,P,0.001 for all subjects). At 15 mM concentration of glucose,baseline insulin values before stimulation with arginine werenot statistically significantly different between the threegroups. There was a significant inverse correlation betweenglucose infusion rate and AIR to arginine at normoglycemiclevels (r520.63, P50.006) (Fig. 4). After stimulation witharginine (Table 3B), although the absolute levels were higherin both the transplanted groups as compared with controls,

the percentage of basal insulin secreted after stimulationwith arginine at hyperglycemia was smaller in the trans-planted groups than in the normal subjects (1191% in thecontrol group as compared with 754% (P,0.03) and 644%(P,0.02) in the FK-506 group and CsA group, respectively).When values for C-peptide were compared, no statisticalsignificant differences were observed before and after thestimulation with arginine at normoglycemia and at 15 mM.

a cell function. At baseline values of glucagon were similarin the three groups. At normoglycemia, the AGR to arginine

FIGURE 4. Correlation between acute insulin response and total glu-cose disposal at fasting glucose levels including all the patients in thestudy except three patients from a total of eight that belong to thecontrol group for which data were not available.

TABLE 3. Basal levels and acute response to arginine of plasma insulin, C-peptide, and glucagon at normo- and hyperglycemiaa

a A, Basal levels before arginine stimulation at 5.5 and 15 mM glucose, plasma insulin, C peptide, and glucagon. B, Acute response ofplasma insulin (AIR), C peptide (ACR), and glucagon (AGR) to arginine at 5.5 mM and at 15 mM of glucose for recipients of liver

transplants treated with FK-506, CsA as well as the control group.

FK-506 CsA Control P(FK-506 vs. control)

P(CsA vs. control)

A. 5.5 mM glucoseP-Insulin (pmol/L) 32.564.9 23.264.5 24.267.1 NS NSP-C-Peptide (pmol/L) 412696 321681 275695 NS NSP-Glucagon (pmol/L) 22.661.2 22.262.8 16.762.8 NS NS

At 15 mM glucoseP-Insulin (pmol/L) 156645 175639 95645 NS NSP-C-Peptide (pmol/L) 16066448 17516364 10696389 NS NSP-Glucagon (pmol/L) 17.661.6 18.262.3 15.460.7 NS NS

FK-506 CsA Control

AUC (2–5 min) % Baseline AUC (2–5 min) % Baseline AUC (2–5 min) % Baseline

B. At 5.5 mM glucoseAIR (pmol/L) 923.46198 645695a 5986181 5486203b 3596189 306652ACR (pmol/L) 29586677 275633 20246297 230625 25036711 238649AGR (pmol/L) 140619 189613 130632 190615 136635 2256100

At 15 mM glucoseAIR (pmol/L) 43926793 7546100c 47826958 6446102d 409262931 11916174ACR (pmol/L) 1389763515 220616 615261197 194633 1221862196 286632AGR (pmol/L) 101620 190630 92613 17869 71634 181618

Values are means 6SEM.a FK-506 versus control, P,0.01.b CsA versus control, P,0.03.c FK-506 versus control, P,0.03.d CsA versus control, P,0.02.

FIGURE 3. Correlation of acute insulin response to i.v. arginine atfasting glucose including all the patients in the study except threepatients from a total of eight that belong to the control group forwhich data were not available.

FERNANDEZ ET AL.November 27, 1999 1537

was 1.89 times higher as compared to the baseline levels inthe FK-506 group, 1.9 times higher in the CsA group, and2.25 times higher in the control group (NS). At hyperglycemiclevels, prestimulus glucagon levels decreased 22% in theFK-506 group, 18% in the CsA group, and only 8% in thecontrol group (NS). However, the AGR to arginine at hyper-glycemia (15 mM) failed to decline as much as in the FK-506group (28%), CsA group (29%) as compared to the controlgroup (48%) (P,0.05) (Table 4).

Cholesterol, triglycerides high density lipoprotein (HDL)and low density liopoprotein (LDL) levels. Total cholesterol,triglyceride, and HDL levels were no different in the FK506,CsA, and control group (TC: 3.660.4, 4.160.5, and 4.260.1,TG: 1.260.2, 1.660.2, and 1.260.3, HDL: 0.960.03, 1.060.2,and 1.060.1 mM, respectively). LDL levels, however, werelower in the FK506 group (1.760.3 mM) as compared tocontrols (2.760.1 mM) (P,0.01) or CsA (2.460.5 mM) (NS).

DISCUSSION

The long-term metabolic complications after renal trans-plantation are well known in the literature, whereas few dataexist regarding the prevalence of metabolic complicationsafter liver transplantation (1). Diabetes mellitus frequentlydevelops de novo after liver transplantation. Although thiscomplication is usually transient, in some patients it hasbeen described as a permanent complication, most likelyrelated to the use of immunosuppressive drugs. In view of thecontradictory reports on the long-term metabolic effects ofcurrent immunosuppressive drugs after liver transplanta-tion (9, 11–13, 20, 21, 25, 26), our study attempts not only toelucidate the differences between these two drugs (comparedwith a normal control population), but also to define thecontributing mechanisms, that occur in vivo, responsible forthe development of PTDM.

The use of FK-506 or cyclosporin A has been associatedwith an incidence of diabetes mellitus up to 40%. Recentstudies suggest that the mechanism of action is similar forboth drugs by preventing Ca11/calmodulin activation of cal-cineurin (14) and thus reversibly inhibit insulin gene tran-scription, leading to a decline in insulin mRNA levels, insulinsynthesis, and ultimately insulin secretion (14). However,FK-506 has been shown to be 10 to 100 times more potentthan CsA and a clear dose dependency of metabolic sideeffects has been demonstrated. The steroid sparing effects ofFK506 is also known (22), perhaps associated to their steroidreceptor binding capacity (FK-FKBP) which decreases theneed for high amount of steroids (44). Most of the studiesaddressing the diabetogenic side effects of FK506 or CsA areconfounded by high maintenance doses of steroids. Steroidsexert their diabetogenic effect not only by increasing hepaticglucose production via stimulation of gluconeogenesis, by anincreased insulin resistance, and consequently hyperinsulin-

emia (45), but also, as recently reported, by directly inhibit-ing insulin secretion (6), and by affecting the metabolism ofFFA (free fatty acids) in liver transplanted patients (54). Toeliminate or reduce these confounding effects of steroid treat-ment, we studied patients in a clinical setting who were instable condition, approximately 1 year after orthotopic livertransplantation with prednisolone doses of #4 mg on eitherFK506 or CsA. These patients were well matched with regardto age, sex, body composition, and follow-up after transplan-tation to allow for the comparison of insulin sensitivity, in-sulin secretory response, a cell function, as well as lipidmetabolism between the FK506 and CsA group.

The results of our study showed that baseline plasma in-sulin levels during the normo- and hyperglycemic clampstudies were not significantly different between the FK506when compared with the CsA group. There was, however, asignificant difference between the controls and the immuno-suppressed groups in terms of total glucose disposal, suggest-ing a higher insulin resistance in the FK506- and CsA-treated patients. It has been previously reported that afterliver transplantation patients have a lower fasting glucoseand hepatic glucose production as compared to patients witha similar immunosuppressive regimen or normal controls(46). In our study hepatic glucose production was similar atbaseline in all three groups but almost completely inhibitedduring the hyperinsulinemic clamp in the liver transplantgroups, whereas in the control group there was a higherhepatic glucose production during the clamp study. Also,differences in terms of nonoxidative glucose metabolism be-tween the transplanted group as compared with the controlgroup correspond to the higher insulin resistance observed inthe transplanted groups. Higher insulin resistance is associ-ated with a lower nonglucose oxidative metabolism. More-over, during euglycemic-hyperinsulinemia, the inhibition offree fatty acid concentration was significantly higher in thecontrol group when compared with the transplanted group. Itis unknown whether the changes observed in glucose andfree fatty acid metabolism are transient changes that occurin the posttransplant period or are permanent changes thatwill contribute to the development of PTDM. In previousreports, it has been demonstrated that after liver transplan-tation there is a gradual return to normalization of insulin-dependent glucose metabolism over a 2-year period (47).

When evaluating b cell secretory capacity, the absoluteacute insulin response to arginine at normoglycemic levelswere higher in the FK506 and CsA group as compared withthe normal controls but not at statistically significant levels.However, the percentage of insulin above basal were allstatistically significant different when compared the trans-planted patient in both groups when compared with controls.The strong inverse correlation between insulin sensitivityand acute insulin response to arginine in all three groups

TABLE 4. a cell function at baseline and after arginine stimulation during normo- and hyperglycemia

Baselineglucagon (pmol/L)

Incremental AUC (2–5 min) for glucagon after argininestimulation

5.5 mMGlucose

15 mMGlucose

%Decrement

P(vs. control) 5.5 mM 15 mM %

DecrementP

(vs. control)

FK-506 22.661.2 17.661.6 22.1 NS 140619 101620 27.9 ,0.05CsA 22.262.8 18.262.3 18.0 NS 130632 92613 29.2 ,0.05Controls 16.762.8 15.460.7 7.8 136635 71634 47.8

TRANSPLANTATION1538 Vol. 68, No. 10

might explain the difference obtained between the trans-planted groups and the normal controls with regard to abso-lute and relative increases after arginine stimulation (Fig. 4).Patients with lower total glucose disposal, i.e., patients withhigher insulin resistance show a greater increase of insulinsecretion upon b cell stimulation with arginine. In contrast,those patients with higher insulin sensitivity, show a lesspronounced insulin release in response to the same stimulus.To detect subclinical abnormalities in beta cell reserve weused the submaximal acute insulin response during glucosepotentiation of arginine-induced insulin secretion at 15 mMglucose as a sensitive indicator of beta cell secretory dysfunc-tion (48). The b cell secretory reserve was statistically differ-ent between FK-506- and CsA-treated patients as comparedto normal controls, despite of the higher absolute values inthe transplanted group. These results are in accordance withprevious studies in recipients of segmental and whole pan-creas transplants (49). The decrease in the secretory reservefound in both transplanted groups might result in an insuf-ficient insulin secretion during periods of increased demandon the b cell and, thus could contribute to the development ofposttransplant diabetes mellitus. However, no differencescould be observed between the two immunosuppressive drugsat least at maintenance doses, in contrast to multiple publi-cations in the past who showed a higher diabetogenic effect ofFK506 compared with CsA (11, 16). In our study where bothimmunosuppressive regimens proved to be equally diabeto-genic, the steroid-sparing effect of FK506 and the lower tri-glyceride and LDL-cholesterol levels (44) might be beneficialin the long-term follow-up and may lead to a reduction incardiovascular and neurovascular complications commonlyseen 5–10 years after solid organ transplantation (11).

In the patients included in our study, statistically signifi-cant differences in creatinine clearance were demonstratedin both transplanted groups as compared to the controlgroup. Previous studies showed a 30 to 40% reduction increatinine clearance compared with baseline levels beforetransplant (20, 50, 51). C-peptide measurements are difficultto evaluate, because of the prolonged half-life of C-peptidedue to the decreased creatinine clearance (52).

The similar baseline glucagon levels at normoglycemia andin response to arginine in all groups and the impaired de-crease in the glucagon levels in response to arginine at hy-perglycemia in both transplanted groups as compared withnormal patients, indicate a normal a cell response at normo-glycemic levels, but a decreased response to the inhibitoryeffect of hyperglycemia during arginine stimulation at 15mM glucose levels. Our results confirm previous findingsthat the increase in glucagon release during arginine stimu-lation is apparently reduced by hyperinsulinemia in normalsubjects (55, 56). A deterioration of this normal response waspreviously studied after segmental pancreas transplant ascompared with whole organ transplantation. It was postu-lated that this defect was secondary to an insufficient trans-planted pancreatic mass in recipients of segmental pancreaswithout considering a possible synergistic deleterious effectof the immunosuppressive agents per SE. In our study, wedemonstrate a damage of the b cell-a cell axis, which mostlikely is induced by the use of immunosuppressive agents. Toour knowledge, this is the first report demonstrating a re-duced inhibition of glucagon secretion during hyperglycemic,hyperinsulinemic conditions in immunosuppressed whole or-

gan recipients with intact pancreatic function. This damageof the b cell-a cell axis is most likely induced by the use ofimmunosuppressive agents.

Protein and lipid oxidation. Prednisone and CsA havebeen shown to affect protein metabolism both in vitro as wellas in vivo (53–55). It has been recently demonstrated, how-ever, that the effect on protein metabolism measured bymeans of restoration of normal basal leucine and leucineoxidation flux is independent of the amount of prednisoneafter liver transplantation. Liver denervation was suggestedas an explanation for this defect (54). This difference ob-served 1 year after liver transplantation might represent atransiently altered insulin-stimulated leucine turnover thatoccurs after liver transplantation and normalizes approxi-mately 2 years posttransplant (54) In our study there was astatistical higher protein oxidation in patients treated withFK506 as compared with normal controls and patientstreated with CsA, suggesting a more pronounced influence ofFK506 as compared to CsA on protein oxidation, independentof the steroid dose. This effect may be offset by the steroidsparing effect of FK506.

Lipid levels. The total cholesterol and high density choles-terol levels were not different between the three groups norwas the cholesterol to HDL ratio. Triglycerides showed atrend to being higher in the CsA as compared with the FK506group and LDL levels were lower in the FK506 group. Thesefindings are in accordance with previous reports (44, 53) thatshowed significantly higher triglyceride levels in CsA-treatedpatients, but no effect on cholesterol, LDL, or HDL, whereasFK506 was associated with a moderate reduction in choles-terol and LDL, but did not affect triglycerides and HDL.Because posttransplant hyperlipidemia and diabetes melli-tus represent a major risk factor for cardiovascular morbidityand mortality (54), an immunosuppressive regimen that min-imizes these risk factors is preferable.

Because the protocol used in our institution favors a fastreduction of steroids and immunosuppressive drugs, we wereable to perform those studies at FK506 and CsA levels withvirtually no maintenance doses of steroids 1 year after livertransplantation. Our results, therefore, are representative ofthe metabolic side effects that can be expected in the long-term follow-up after liver transplantation. To our knowledgethis is also the first study to compare the immunosuppressiveregimen of FK506 with CsA on low-maintenance doses with-out the confounding effects of high or different steroid levelsin the two groups. This study also confirms previous reportsthat complete steroid withdrawal is safe and immunologi-cally tolerated by liver transplant recipients (1, 55). Thereexist numerous reports that show a strong association ofdiabetes mellitus and chronic hepatitis C (56–60) that is notpresent in patients with chronic hepatitis B. Although weexcluded patients with diabetes from the study, the selectionof hepatitis C patients represents a high risk cohort fordiabetes development. Therefore, this seems to be an idealgroup to study the diabetogenic effects of FK506 and cyclo-sporine A, because these patients might have minor changesin glucose metabolism that augment the diabetogenic poten-tial of these drugs. Contrary to some publications that re-ported a more pronounced diabetogenicity of FK506 as com-pared to CsA we found no major difference in insulinsensitivity and insulin secretory reserves between the twogroups. On the contrary, the trend toward lower maximum

FERNANDEZ ET AL.November 27, 1999 1539

insulin secretion and higher TG and LDL levels in CsA ascompared with FK506 might prove beneficial in hyperlipid-emia or situations with a reduced or limited b cell mass andinsulin resistance such as impaired glucose tolerance beforetransplantation or after segmental or islet transplantation.The significant insulin resistance and impaired secretorycapacity of the b cell in the transplanted groups as opposed tonormal controls, as well as the defect in b cell-a cell axis arefactors that contribute to the development of PTDM in agroup of patients at high risk for diabetes development due tochronic hepatitis C infection (56–60).

REFERENCES

1. Stegall MD, Everson G, Schroter G, Bilir B, Karrer F, Kam I.Metabolic complications after liver transplantation. Diabetes,hypercholesterolemia, hypertension, and obesity. Transplanta-tion 1995; 60: 1057.

2. Navasa M, Bustamante J, Marroni C, et al. Diabetes mellitusafter liver transplantation: prevalence and predictive factors.J. Hepatol. 1996; 25: 64.

3. Maser RE, Wolfson SK Jr, Ellis D, et al. Cardiovascular diseaseand arterial calcification in insulin- dependent diabetes melli-tus: interrelations and risk factor profiles. Pittsburgh Epide-miology of Diabetes Complications Study. Arterioscler Thromb1991; 11: 958.

4. Trail KC, Stratta RJ, Larsen JL, et al. Results of liver trans-plantation in diabetic recipients. Surgery 1993; 114: 650.

5. Rizza RA, Mandarino LJ, Gerich JE. Effects of growth hormoneon insulin action in man. Mechanisms of insulin resistance,impaired suppression of glucose production, and impairedstimulation of glucose utilization. Diabetes 1982; 31: 663.

6. Delaunay F, Khan A, Cintra A, et al. Pancreatic beta cells areimportant targets for the diabetogenic effects of glucocorti-coids. J Clin Invest 1997; 100: 2094.

7. Sumrani NB, Delaney V, Ding ZK, et al. Diabetes mellitus afterrenal transplantation in the cyclosporine era—an analysis ofrisk factors. Transplantation 1991; 51: 343.

8. Kahan BD, Flechner SM, Lorber MI, Golden D, Conley S, VanBC. Complications of cyclosporine-prednisone immunosup-pression in 402 renal allograft recipients exclusively followedat a single center for from one to five years. Transplantation1987; 43: 197.

9. Krentz AJ, Dousset B, Mayer D, et al. Metabolic effects of cyclo-sporin A and FK 506 in liver transplant recipients. Diabetes1993; 42: 1753.

10. Ericzon BG, Wijnen RM, Kubota K, Kootstra G, Groth CG.FK506-induced impairment of glucose metabolism in the pri-mate—studies in pancreatic transplant recipients and in non-transplanted animals. Transplantation 1992; 54: 615.

11. Jindal RM, Popescu I, Schwartz ME, Emre S, Boccagni P, MillerCM. Diabetogenicity of FK506 versus cyclosporine in livertransplant recipients. Transplantation 1994; 58: 370.

12. Strumph P, Kirsch D, Gooding W, Carroll P. The effect of FK506on glycemic response as assessed by the hyperglycemic clamptechnique. Transplantation 1995; 60: 147.

13. Scantlebury V, Shapiro R, Fung J, et al. New onset of diabetes inFK 506 vs cyclosporine-treated kidney transplant recipients.Transplant Proc 1991; 23: 3169.

14. Redmon JB, Olson LK, Armstrong MB, Greene MJ, RobertsonRP. Effects of tacrolimus (FK506) on human insulin gene ex-pression, insulin mRNA levels, and insulin secretion in HIT-T15 cells. J Clin Invest 1996; 98: 2786.

15. Robertson RP. Cyclosporin-induced inhibition of insulin secre-tion in isolated rat islets and HIT cells. Diabetes 1986; 35:1016.

16. Ishizuka J, Gugliuzza KK, Wassmuth Z, et al. Effects of FK506

and cyclosporine on dynamic insulin secretion from isolateddog pancreatic islets. Transplantation 1993; 56: 1486.

17. Krausz Y, Wollheim CB, Siegel E, Sharp GW. Possible role forcalmodulin in insulin release. Studies with trifluoperazine inrat pancreatic islets. J Clin Invest 1980; 66: 603.

18. Liu J, Farmer JDJ, Lane WS, Friedman J, Weissman I, Schre-iber SL. Calcineurin is a common target of cyclophilin-cyclo-sporin A and FKBP-FK506 complexes. Cell 1991; 66: 807.

19. Gillison SL, Bartlett ST, Curry DL. Inhibition by cyclosporine ofinsulin secretion–a beta cell-specific alteration of islet tissuefunction. Transplantation 1991; 52: 890.

20. The U.S.Multicenter FK506 Liver Study Group. A comparison oftacrolimus (FK 506) and cyclosporine for immunosuppressionin liver transplantation. N Engl J Med 1994; 331: 1110.

21. European FK506 Multicentre Liver Study Group. Randomisedtrial comparing tacrolimus (FK506) and cyclosporin in preven-tion of liver allograft rejection. Lancet 1994; 1: 423.

22. Ricordi C, Zeng YJ, Alejandro R, et al. In vivo effect of FK506 onhuman pancreatic islets. Transplantation 1991; 52: 519.

23. Hirano Y, Fujihira S, Ohara K, Katsuki S, Noguchi H. Morpho-logical and functional changes of islets of Langerhans inFK506-treated rats. Transplantation 1992; 53: 889.

24. Steinmuller TM, Graf KJ, Schleicher J, et al. The effect of FK506versus cyclosporine on glucose and lipid metabolism—a ran-domized trial. Transplantation 1994; 58: 669.

25. Mieles L, Gordon RD, Mintz D, Toussaint RM, Imventarza O,Starzl TE. Glycemia and insulin need following FK 506 rescuetherapy in liver transplant recipients. Transplant Proc 1991;23: 949.

26. Tabasco-Minguillan J, Mieles L, Carroll P, Gavaler J, Van TD,Starzl TE. Insulin requirements after liver transplantationand FK-506 immunosuppression. Transplantation 1993; 56:862.

27. The Expert Committee on the Diagnosis and Classification ofDiabetes Mellitus. Report of the expert committee on the di-agnosis and classification of diabetes mellitus. Diabetes Care1997; 20: 1183.

28. Luzi L, Secchi A, Facchini F, et al. Reduction of insulin resis-tance by combined kidney-pancreas transplantation in type 1(insulin-dependent) diabetic patients. Diabetologia 1990; 33:549.

29. Luzi L, Hering BJ, Socci C, et al. Metabolic effects of successfulintraportal islet transplantation in insulin-dependent diabetesmellitus. J Clin Invest 1996; 97: 2611.

30. Thiebaud D, Jacot E, DeFronzo RA, Maeder E, Jequier E, FelberJP. The effect of graded doses of insulin on total glucose up-take, glucose oxidation, and glucose storage in man. Diabetes1982; 31: 957.

31. Castellino P, Luzi L, Simonson DC, Haymond M, DeFronzo RA.Effect of insulin and plasma amino acid concentrations onleucine metabolism in man. Role of substrate availability onestimates of whole body protein synthesis. J Clin Invest 1987;80: 1784.

32. Ferrannini E. The theoretical bases of indirect calorimetry: areview. Metabolism 1988; 37: 287.

33. Palmer JP, Walter RM, Ensinck JW. Arginine-stimulated acutephase of insulin and glucagon secretion. I. in normal man.Diabetes 1975; 24: 735.

34. DeFronzo RA, Tobin JD, Andres R. Glucose clamp technique: amethod for quantifying insulin secretion and resistance. Am JPhysiol 1979; 237: E214.

35. Wielko J, Sherman WR. Boroacetylation of carbohydrates. Cor-relations between structures and mass spectral behavior inmonoacetylhexosis cyclic boronic esters. J Am Chem Soc 1976;98: 7631.

36. Luzi L, Castellino P, DeFronzo RA. Insulin and hyperaminoaci-demia regulate by a different mechanism leucine turnover andoxidation in obesity. Am J Physiol 1996; 270: E273.

TRANSPLANTATION1540 Vol. 68, No. 10

37. Steele R. Influence of glucose loading and of injectd insulin onhepatic glucose output. Ann N Acad Sci 1959; 82: 420.

38. Frayn KN. Calculation of substrate oxidation rates in vivo fromgaseous exchange. J Appl Physiol 1983; 55: 628.

39. Hawk PD. Kjeldahl method. In: Practical physiological chemis-try. pp 814–822. Toronto: Blakiston, 1947.

40. Ferrannini E, Natali A, Brandi LS, et al. Metabolic and thermo-genic effects of lactate infusion in humans. Am J Physiol 1993;265: E504.

41. Halter JB, Graf RJ, Porte D Jr. Potentiation of insulin secretoryresponses by plasma glucose levels in man: evidence that hy-perglycemia in diabetes compensates for imparied glucose po-tentiation. J Clin Endocrinol Metab 1979; 48: 946.

42. Ward WK, Bolgiano DC, McKnight B, Halter JB, Porte D Jr.Diminished B cell secretory capacity in patients with noninsu-lin-dependent diabetes mellitus. J Clin Invest 1984; 74: 1318.

43. Ward WK, Wallum BJ, Beard JC, Taborsky GJJ, Porte D Jr.Reduction of glycemic potentiation. Sensitive indicator of beta-cell loss in partially pancreatectomized dogs. Diabetes 1988;37: 723.

44. Jindal RM, Popescu I, Emre S, et al. Serum lipid changes in livertransplant recipients in a prospective trial of cyclosporine ver-sus FK506. Transplantation 1994; 57: 1395.

45. McMahon M, Gerich J, Rizza R. Effects of glucocorticoids oncarbohydrate metabolism. Diabetes Metab Rev 1988; 4: 17.

46. Perseghin G, Regalia E, Battezzati A, et al. Regulation of glucosehomestasis in humans with denervated livers. J Clin Invest1997; 99: 931.

47. Luzi L, Perseghin G, Regalia E, et al. Metabolic effects of livertransplantation in cirrhotic patients. J Clin Invest 1997; 99:692.

48. Teuscher AU, Seaquist ER, Robertson RP. Diminished insulinsecretory reserve in diabetic pancreas transplant and nondia-betic kidney transplant recipients. Diabetes 1994; 43: 593.

49. Christiansen E, Tibell A, Volund A, et al. Pancreatic endocrinefunction in recipients of segmental and whole pancreas trans-plantation. J Clin Endocrinol Metab 1996; 81: 3972.

50. De Mattos Am, Andoh TF, Bennett WM. Cyclosporine nephrop-

athy: clinical, histological, and functional aspects. TransplantRev 1996; 10: 225.

51. Klintmalm G. A review of FK506: a new immunosuppressantagent for the prevention and rescue of graft rejection. Trans-plant Rev 1994; 8: 53.

52. Blackman JD, Polonsky KS, Jaspan JB, Sturis J, Van Cauter E,Thistlethwaite JR. Insulin secretory profiles and C-peptideclearance kinetics at 6 months and 2 years after kidney-pan-creas transplantation. Diabetes 1992; 41: 1346.

53. Hohage H, Arlt M, Bruckner D, Dietl KH, Zidek W, Spieker C.Effects of cyclosporin A and FK 506 on lipid metabolism andfibrinogen in kidney transplant recipients. Clin Transplant1997; 11: 225.

54. Vathsala A, Weinberg RB, Schoenberg L, et al. Lipid abnormal-ities in cyclosporine-prednisone-treated renal transplant recip-ients. Transplantation 1989; 48: 37.

55. Padbury RT, Gunson BK, Dousset B, et al. Steroid withdrawalfrom long-term immunosuppression in liver allograft recipi-ents. Transplantation 1993; 55: 789.

56. Mason AL, Lau JY, Hoang N, et al. Association of diabetesmellitus and chronic hepatitis C virus infection. Hepatology1999; 29: 328.

57. Knobler H, Stagnaro GA, Wallenstein S, Schwartz M, RomanSH. Higher incidence of diabetes in liver transplant recipientswith hepatitis C. J Clin Gastroenterol 1998; 26: 30.

58. Grimbert S, Valensi P, Levy MC, et al. High prevalence ofdiabetes mellitus in patients with chronic hepatitis C. A case-control study. Gastroenterol Clin Biol 1996; 20: 544.

59. Fraser GM, Harman I, Meller N, Niv Y, Porath A. Diabetesmellitus is associated with chronic hepatitis C but not chronichepatitis B infection. Isr J Med Sci 1996; 32: 526.

60. Allison ME, Wreghitt T, Palmer CR, Alexander GJ. Evidence fora link between hepatitis C virus infection and diabetes melli-tus in a cirrhotic population. J Hepatol 1994; 21: 1135.

Received 7 December 1998.Accepted 20 May 1999.

FERNANDEZ ET AL.November 27, 1999 1541