Conte, D

Francavilla, A

Kam, I

Lynch, S

Polimeno, L

Reale, L

Starzl, TE

Tedane, E

Todo, S

Van Thiel, DH

English

Name not available

Insulin, glucagon, peptide and amino acid levels following liver transplantation

D-Scholarship@Pitt

Insulin, Glucagon, C-Peptide and Aminoacid Levels Following Liver Transplantation A. Francavilla l , L. Polimeno l , D.H. Van ThieF, T. Saturo3 , I. Kam3 , S. Lynch3 , L. Reale l , E. Tedone l , D. Conte l and T.E. StarzP I Dept. of Gastroenterology, University of Bari, Bari, Italy 2 Dept. of Gastroenterology and 3 Dept. of Surgery, University of Pittsburgh, Health Center, Pittsburgh, Pennsylvania INTRODUCTION Glucose intolerance (5,14), hyperinsulinemia, (3,9), insulin-resistance (2,4) and hyperglucagonemia (12,17,18) are common in patients with advanced liver disease. Several hormonal abnor-malities are reported in cirrothic patient and one thought to eighte~ contribute to or be responsible for the reduced plasma concentrations of the three principal branched chain acids (BCAA) valine, leucine and isoleucine that are characteristic of end-stage liver disease. As' a result, these same hormonal abnor-malities are thought also to contribute to the decreased molar ratio that exists in plasma between the BCAA and the aromatic amino acids (AAA) tyrosine, phenylalanine and tryptophan in cir-rhotics (7,13,15,20). We report our observations concerning the plasma levels of insulin and glucagon and the major aminoacids before and after orthotopic liver transplantation (OLTx). The changes observed occur in cirrhotics postoperatively were compared to those observed to occur in a non-cirrhotic human recipient with normal liver function, who underwent liver transplantation for multiple hepatic adenomatosis and to those observed in five normal dogs which were submitted to liver replacement under controlled laboratory conditions. 56 HORMONES AND LIVER TRANSPLANTATION MATERIAL AND METHODS Case Material The clinical characteristics of the eight cirrhotic patients and the single individual with multiple hepatic adenomatosis studied ere shown in Table l. As can be seen from the Table, patient with multiple hepatic adenomatosis had normal function. TABLE 1: PATIENTS TREATED WHITB OLTx NAME SEX AGE DIAGNOSIS BILIRUBIN PROTHROMBIN ALBUMIN (years) (mg/l00m1) Time (sec) (g/ll BR M IS Chronic active hepatitis 3S.S lS/13 2.7 RY F 25 Chronic active hepatitis 9.9 15/13 2.6 TH F 23 Chronic active hepatltis 2.2 16/13 2.2 GH M 37 Sclerosing Cholangitis 19.7 15/13 2.6 SJ F 41 Sclerosing Cholangitis 15.3 14.4/13 2.9 KD M 34 Sclerosing Cholangitis 23.7 14/13 2.8 SR F 29 Wilson's Disease 3.5 16.S/13 2.7 SF F 43 1 Antitrypsin deficency 4.9 14/13 2.8 FOR NEOPLASM GB F 29 Adenomatosis 0.5 13/13 3.0 Clinical characteristics, bilirubin, prothrombin time and albumin levels of the eight cirrhotic patien~s and the single patient with multiple adenomatosis. the liver In contrast, the patients with liver disease all had moderately advanced liver disease as documented by the abnormalities in their blirubin, prothrombin time and albumin levels. Cyclosporine and prednisone were used as the immunodepressive agents for all of the recipients studied (21,22). The five mongrel dogs transplanted and studied weighted between 15-20 kg and like the human studied, were given cyclosporine and prednisone to prevent rejection using doses that, on a mg per kg basis, were similar to those used by the patients (10). The surgi-cal proc~dures used for the patients and animals was identical (10,22) . Special Analysis The plasma levels of immunoreactive insulin (IRI) were deter-mined by radioimmunoassay using an insulin kit obtained from Serono Diagnostics (Braintree, MO). The detection limit of the as-say was 5 U/ml. The plasma levels of C-peptide (CP) were deter-mined by radioimmunoassay using a C-peptide kit obtained from Serono Diagnostics.The detection limit of the assay was 0.2 ng/ml. HORA The plasma le' mined with a gluc This kit was c accuracy, precis et al (11). Sa chilled tubes c (aprotinin) and for assay. The detection For representa studied, both be technique (1), w glucagon having Insulin/glucaf Muller (16) usi Ratio. This formu 6.000 D and a b that glucagon has cyclosporine, to postoperatively, was found not to Amino acid pro plasma treated wi natants were Instruments, Some. cluding tryptoph were made using a The unpaired S of the data. A p Glucagone, In!' patients studied. normal preoperat hours following 1 ng/ml and remaini The plasma I (Fig. LA). Fall crease in the I IN ,tic patients . adenomatosis Ie Table, the normal liver IN ALBUMIN .: I (gill 2.7 2.6 2.2 2.6 2.9 2.8 2.7 2.8 3.0 albumin lent with had moderately ities in their :munodepressive dghted between :losporine and on a mg per kg LO). The surgi-> was identical were deter-t obtained from imit of the as-CP) were deter-obtained from , was 0.2 ng/ml. HORMONES AND LIVER TRANSPLANTATION 57 The plasma levels of immunoreactive glucagon (IRG) were deter-mined with a glucagon kit also obtained from Serono Diagnostics. This kit was chosen specifically because of its high degree of accuracy, precision and specificity as has been reported by Kenny et al (11). Samples for the glucagon assay were collected in chilled tubes containing 500 units of a trypsin inhibitor (aprotinin) and 1.2 mg of sodium EDTA/ml of whole blood collected for assay. The detection limit for the assay was 15 pg/ml. For representative blood samples obtained from each patient studied, both before and after transplantation, a gel filtration technique (1), was used to determine the amount of IRG-detectable glucagon having molecular weights of 3.500, 7.000 and 40.000 D . Insulin/glucagon molar rations were calculated as suggested by Muller (16) using the formula (IRI (uU/ml)/IRG (pg/ml) x 23.33 = Ratio. This formula assumes that insulin has a molecular weight of 6.000 D and a biologic activity of 25 units/mg. It also assumes that glucagon has a molecular weight of 3,500 D. The addition of cyclosporine, to achieve concentration of the drug found in blood postoperatively, to samples of blood assayed for IRG, CP, and IRI, was found not to effect the assay results. Amino acid profiles were determined on deproteinized samples of plasma treated with 4% sulfosalicylic acid. The resultant super-natants were applied to an amino acid analyzer (Beckman Instruments, Somerset, NJ) and the levels of free BCAA and AAA in-cluding tryptophan were determined: The glucose determinations were made using a glucose-oxidase method (8). Statistical Analysis The unpaired Student's t-test was used for statistical analysis of the data. A p value <0.05 was considered to be significant. RESULTS End-Stage Liver Disease Glucagone, Insulin and C-peptide levels (Fig. LA). In the eight patients studied, the C-peptide plasma concentrations were allhigh normal preoperatively and rose 2 and 1/2-fold during the first 12 hours following liver transplantation to a maximum level of 10 ng/ml and remaining elevated for a least 30 days (Fig. lA). The plasma IRI levels followed a somewhat different pattern (Fig. LA). Following transplantation, there was a two-fold in-crease in the IRI levels but this increase lasted only for a new 58 HORMONES AND LIVER TRANSPLANTATION hours and subsequently returned to near normal levels at four days post-transplantation. 121 IU 10 !:!- 8 ~! 8 "", Q.c 4 ,-" 0 .. A r± --g±i~ " , I'!I 20 TIME AFlU aLT. (dOYI) B KDjI~ ~ { 'OJ i~ o 2", i 20 ~ 10 • 0 10 1!1 20 nME .. .FlER OlT. (da,,1t When studied at six months, both the insulin and C-peptide plasma levels were slightly elevated (Table 2). TABLE 2: LEVELS OF GLUCAGON, INSULIN, C-PEPTIDE AND IRI/IRG MOLAR RATIO NORMAL SUBJECTS CIRRHOTIC PATIENTS Before OLTx 30 daYS ai'ter OLTx 180 days after OLTx tpTAL , GLUCAGON (pg/ml) 10, 90,!lS 35O.!, 60 P 0.05 e ·750.=110 p 0.01 eo! 12 n.s. M.IM. 3500 GLUCAGON (pg/ml) 75Z16 294.!.51 P 0.05 638=.87 p' 0.01 68=.9 n.s. INSULIN C-PEPTIDE IRI/IRG (uUIImll (ng/ml) 25!.5 4 .::0.5 1. 5.!O. 5 n.s. n.S. p 0,01 22.:.8 6 .::1 O.6!,O.1 • n.s. p 0.01 P 0.001 30.:7.15 3.8!,O.5 10.2,:1 p 0.05 n.s. p 0.001 Levels of glucagon, C-peptlde. insulin Rnd IRIIIRG ratio in nOI',!,al subJects, and in 8 cirrhotic patients who underwent liver transplantation. The deterMinations in the operated patients were made before transplantation, 30 days and 180 days after transplantation. The data for "normal subjects" refer to deterMlnations obtuned 1n 10 healthy people age 18-45 years. Each value is express~d as M + S.D. Significance (p) 1n relation to the values of normal subjects. HG Glucagon - r times the norm<J level rose prof were found at when restudied 2). The result; as a conseque: mal insulin Ie' days. The gel hyperglucagone: predominantly of 3,500 D. T biologically glucose levels glucocorticoid transplantatio 6 days after servation peri Amino Acids the depressed range for ea importantly, (Fig. IB) sub 30-day study p, \ at four days B . ,. ~rllh and C-peptide IIRG .:0.5 .01 .:0.1 ),001 t5, and ations :0 days , ':lons ,sM;,S.O. HORMONES AND LIVER TRA NSPLA NTA TlON 59 Glucagon - Before operation, the plasma levels of IRG were four times the normal values (Table 2). After transplantation, the IRG level rose progressively. The highest mean plasma levels of IRG were found at 30 days following transplantation (Fig. lA), but when restudied at 6 months had returned to the normal range (table 2). The resultant IRI/IRG molar ratio was decreased significantly as a consequence of the observed hyperglucagonemia with near nor-mal insulin levels observed after the first 4 post-transplant days. The gel filtration studies demonstrated that the hyperglucagonemia, both before and after transplantation, was predominantly due to the presence of IRG having a molecular weight of 3,500 D. This form of the hormone is thought to be the most biologically active fraction glucose levels was observed glucocorticoid therapy used (6). A sharp increase of plasma probably related to the intense during the first days following transplantation. Blood glucose levels-returned to the normal range 6 days after operation and remained normal during all of the ob-servation period (data not reported). Amino Acids - The preoperative elevations of AAA (Fig. IB) and the depressed levIes of BCAA (Fig. IB) returned to the normal range for each within 12 hours after transpalntation. More importantly, the plasma amino acid levels and the BCAA/AAA ratio (Fig. IB) subsequently achieved the normal range for the entire 3D-day study period. B IDOGS) ... ... TIME AFTER Ol T. Idoy.) 60 HORMONES AND LIVER TRANSPLANTATION Control study - No changes were observed in the normal levels of IRI, IRG and the plasma amino acid levels following liver transplantation in the one patient with multiple hepatic adenomatosis studied (Fig. 2A). Similarly, no changes were ob-served for these same substances in the five normal dogs submitted to liver transplantation (Fig. 2B). However, this individual and these dogs had no abnormalities preoperatively that might other-wise heve been expected to correct following successful transplantation. Importantly these data suggest that neighter the doses of prednisone used nor the cyclosporine used to prevent rejection contributed to the hormonal and amino acid finding ob-served in the cirrhotic patients studied. DISCUSSION The purpose of this study was to determine if the hormonal ab-normalities which are characteristic of end-stage chronic liver disease can be corrected by orthopic liver transplantation. Both the hyperinsulinemia and the hyperglucagonemia evident preoperatively persisted for some time after successful transplan-tation suggesting that the hypersecretion of these two hormones which characterizes chronic liver disease was not promptly co'r-rected with elimination of the liver disease per see Although the plasma insulin level gradually returned to the normal range within days of the transplant procedure, the plasma glucagon level con tinued to be abnormally elevate and in fact became progressively more abnormal (more elevated) for several weeks before it also be-came normal sometime between 1 and 6 months postoperatively. These data coupled with the data obtained from a patient with multiple hepatic adenoma but normal liver function suggest that the abnor-malities seen in the patients with advanced liver disease reflected alterations in hormone balance and energy metabolism which were due to the individual's advanced liver disease and not to the surgical procedure and the immunotherapy that follow the procedure. These observations also support the prior findings of Sherwin (17, 18) and others (6) that suggest that over production of the pancreatic hormones rather than defective hepatic uptake accounts for the nyperglucagonemia and hyperinsulinemia of ad-vanced liver disease. Even more importantly, the failure to demonstrate any relationship between the changes in the plasma levels of glucagon and either the BCAA or AAA in these studies suggests that the abnormal plasma levels of both classes 'of amino acid present in patients with liver disease are not regulated or determined to a major degree by the hyperglucagonemia present in HOI patients with ad' gest strongly th, advanced liver I tion or maintenal in patients with In contrast sulin levels ret transplant proc the hormone docu and a progress parent increase sient increase transplantation, glucose seen transplanted li\ tal circulatior level of the ho) the hormone by the patien.ts an( not shown), it teration in reno insulin. None t, the increased e-not apparent fr, throughout the , The changes in the eight pa: studied were no tion and multip mal dogs that changes observe appear to be tranplant proce These data; reduced branch. cirrhosis are , in such indivi, lat ter hypote· chronic liver , producing an branched chain (19). The Cll branched chain levels of in~D v IOrmal levels lowing liver i.ple hepatic ~es were ob-'gs submitted ldividual and might other-successful neighter the to prevent J finding ob-'lOrmonal ab--:hronic liver ltion. .emia evident 11 transplan-:wo hormones lromptly co'r-Although the range within level con 'rogressively it also be-ively. These ith multiple the abnor-ver disease metabolism ease and not follow the findings of r production patic uptake mia of ad-failure to the plasma hese studies ;es 'of amino regulated or present in HORMONES AND LIVER TRANSPLANTA T/ON 61 patients with advanced liver disease. Conversely, these data sug-gest strongly that the elevated plasma level of glucagon seen in advanced liver disease plays little important role in the produc-tion or maintenance of the abnormal plasma amino acid levels seen in patients with advanced liver disease. In contrast to the observatios of plasma glucagon, plasma in-sulin levels returned to near normal range within days of the transplant procedure despite apparent persistent hypersecretionof the hormone documented by the increased plasma level of C-peptide and a progressive rise in the C-peptide/insulin ratio. This ap-parent increase in insulin secretion was independent of the tran-sient increase of glucose observed during the first 6 days after transplantation, and is well beyond the period of increased blood glucose seen postoperatively. These data suggest that the transplanted liver actively removes secreted insulin from the por-tal circulation, thereby maintaining a normal systemic plasma level of the hormone despite excess production and secretion of the hormone by the pancreas. Moreover, as the renal function of the patients and the animals studied did not become impaired (data not shown), it is most unlikily that these changes reflect an al-teration in renal handling or clearance of eighter C-peptide or insulin. None the less, it is at least theoretically possible that the increased C-peptide levels reflect cyclosporine nephrotoxicity not apparent from the serial creatinine or BUN determinations made throughout the study. The changes in the plasma levels of insulin and glucagon seen in the eight patients with advanced chronic liver disease who were studied were not seen in the one patient with normal hepatic func-tion and multiple hepatic adenomatosis studied or in the five nor-mal dogs that were trasplanted experimentally. Thus the hormonal changes observed in the patients with advanced liver disease would appear to be specific for liver disease per se and not the tranplant procedure or the immunodepressive regimen used. These data are consistent with the recent suggestion that the reduced branched chain amino acid levels seen in cases of advance cirrhosis are due to the increased plasma levels of insulin seen in such individuals. It has been proposed by the authors of these latter hypotesis that the systemic hyperinsulinemia of advanced chronic liver disease drives plasma ~CAA into adipocytes, thereby producing an abnormal reduction in the plasma level of the branched chain amino acid which is characteristic of cirrhosis (19). The current observations that the plasma levels of the branched chain amino acids normalize concurrently with the plasma levels of insulin provides strong, albeit indirect, support for 62 HORMONES AND LIVER TRANSPLANTATION the hypothesis. Equally important, however, is the observation that the plasma level of branched chain amino acids normalize while the plasma glucagon level becomes progressively more abnormal. This suggests quite strongly that the plasma level of glucagon does not impor-tantly regulate the plasma level of the brancheG chain amino acids. REFERENCES 1. Chisholm DJ., Alford FP., Harewood MS. et al. 1978 Metabolism 27: 262-273. 2. Collins JR., Crofford OB. 1969 Arch. 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Insulin, glucagon, peptide and amino acid levels following liver transplantation

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