Atwater, I

Boschero, AC

Carroll, PB

Gonçalves, AA

Starzl, TE

Tzakis, AG

English

PubMed

Goncalves, AA

Name not available

Effect of the immunosuppressant FK 506 on insulin release from adult rat islets of Langerhans

D-Scholarship@Pitt

In ·1 0-() )r IS )1 Effect of the Immunosuppressant FK 506 on Insulin Release From Adult Rat Islets of Langerhans p.B. Carroll, AA Gongalves, A.C. Boschero, A.G. Tzakis, T.E. Starzl, and I. Atwater CLINICAL introduction of cyclosporine A (CyA) was a critical development that improved the success rate of organ transplantation. I Cy A has also been useful in the elucidation and treatment of type I diabetes as an autoimmune disease in animals2 and humans.) Despite the impressive achievements obtained with the use of CyA. nephrotoxicity has limited its use in an autoimmune dis· ease such as diabetes. Additionally. Cy A decreases insulin synthesis and secretion from several species4•7 and de-creases insulin release from human islets in vitro.8 al-though in vivo glucose tolerance and insulin dynamics may be normal in patients taking the drug.9 FK 506 is a new immunosuppressive agent that is more potent on a weight basis than Cy A 10 and has recently been used successfully in humans undergoing organ transplantations. II Since the drug has important potential applications in the fields of islet, whole pancreas transplantation. and immune inter-vention for type I diabetes. we looked at the effects of FK 506 on insulin secretion from adult rat islets of Langer-hans. METHODS We used freshly isolated islets obtained by collagenase digestion of the pancreas from adult male Wistar rats.12 Insulin was mea-sured by standard radioimmunoassay using a charcoal and dex-tran separation method.1l After isolation, islets were washed live times in basal (2.8 mmollL glucose) containing buffer. We measured insulin released by basal (2.8 or 5.6 mmollL) or stimulatory (8.3 to 22 mmollL) glucose during a 9O-minute static incubation of groups of five freshly isolated islets incubated in the presence or absence of FK S06 (provided by Dr Raman Venka· tarmanan. University of Pittsburgh). Some studies were per· formed using CyA (kindly provided by Sandoz Research Institute. East Hanover. NJ). For all experiments a modified Krebs buffer was used that contained (in mmollL): 120 NaCI. S KCI, 2S NaHCO). 2.5 CaCI1• and LI MgC12• equilibrated with 95% 0.;% CO2, and supplemented with S mglmL bovine serum albumin. pH 7.4. 31"C. Statistics ResuJts are expressed as mean :!: SEM. The statistical significance of insulin release data was analyzed by Student's t test. Data with multiple groups were analyzed using a one-way analysis of vari· ance (ANOY A) and the Wilcoxon ranked sums test. Conclusions drawn from parametric and non parametric analyses were the same. We used a stepwise multiple comparisons procedure to assess FK 506 dose-response results. 14 RESULTS Figure 1 summarizes the effects of acute exposure of islets to various concentrations of either FK 506 or CyA on 120 100 ........ ~ 80 c=::J eo... Z 1: Q 60 !' _ C;oA I--..... I!Zml!I ... -""-0: -u ..... 40 VI 20 -, 0 ,} a 0.1 1.0 10 100 DRUG CONCENTRATION Fig 1. Acute Effect of FK 506 on 16.7 mmoVL glucose-induced insulin release. In this and subsequent figures insulin release was me8SU'ed at the end of a 9O-minute incubation in all conditions. Values are expressed as the mean :t SEM (36 observations). In this and subsequent figures. 100% secretion corresponds to insulin measured in 16.7 mmollL glucose after subtraction of basal release. Basal insulin release (2.8 mmollL glucose-induced) was 650 = 5.8 pglisl",tI9O min, and stimulated release (16.7 mM glucose induced) was 2200.8 ~ 20.9 pglisletl90 rm (P < .01). A concentration of 100 ng/mL FK 506 produced an average inhibition of 35% (P < .05). This concentration was inhbtory in every expennent. In one experiment. a concentration of 10 nglmL was also significantly inhibitory. Comparable data from islets incubated with CyA are shown in filled bars. Stimulated release was 1556.0 :!: 102 pgflSlet!90 min (P < .01) and CyA (1 and 10 #'9/mL) produced a 30% inhibition of this release (P < .OS). glucose-induced insulin secretion. In all experiments 16.7 mmollL glucose evoked a three to sevenfold rise in insulin secretion compared with basal (P < .01). The glucose-stimulated insulin release was inhibited approximately 359C by 100 ng/mL FK 506 (P < .05). This concentration was inhibitory in every experiment. In one experiment 10 ng/mL was also clearly inhibitory From the Laboratory of CeI Biology and Genetics, National Instilule of Diabetes DigestiYe and Kidney Diseases, National Instilutes of Health. Bethesda.. Maryland (P.B.C .• A.A.G., A.C.B .• LA.): and the Department of Surgery, University of Pittsburgh Mecic:al School, Pittsburgh, Pennsylvania (A.G.T •• T.E.S.). A.C.B. was supported by a grant from the Brazilian foundation CAPNS (Grant /I 1662189): AAG. was supported by CNPq, Sao Paolo. Brazil. Address reprint requests to P.B. Carroll, Bldg 8, Room 326. National Institutes of Health. Bethesda. MD 20892. C 1991 by Appleton & Lange Q041-13451911S3'()Ol +0 Transplantation Proceedings, Vol 23. No 1 (February), 1991: pp 337-339 337 338 K~ loor A g 600 " I .. .. '-. 400 '" Q. ! -' '00 ::> <It ! ''0 100 B t' ~ 10 Z e 10 a: ~ '0 III '0 1.8 ~KI ._, 11.2 '6.7 22.2 GLUCOSE (rr#.1) _. Ac::-506 '00 ....... ~ 2. Effect of FK 506 on basal insulin release (A) and glucose dose-response (8). Insulin release in 2.8 mmollL glucose was 624.8 ± 53.0 pglisletl90 min in the control and 616.5 ± 58.3 pglisletl90 min in FK 506-treated islets (n = 60 observations) (P = .92). Data in 8 were normalized to the basal and expressed as a percent of maximal secretion in each condition (n = 18 observa-tions at each glucose concentration. three separate experiments). (P < .05). CyA at concentrations of I and 10 ~mi produced an approximately 30% inhibition of insulin re-lease (P < .05). The effect of I ng/mL FK 506 was not additive with 1 or 10 i4¥mL CyA to further suppress insulin release (data not shown). Figure 2 illustrates the effects of acute exposure of the islets to 100 ng/mL FK 506 on basal-induced (2.8 mmol glucose) insulin release (Fig 2A) or the graded increase in insulin evoked by increasing glucose concentrations (Fig 28). In this series of experiments. basal insulin release was 624.8 ± 53 pg/isletJ90 min and, in the presence of FK 506. was 616.5 ± 58.3 pg/isletl9O min (P = .92). While FK 506 did not inhibit insulin release at lower glucose concentra-tions. it inhibited both 16_7 and 22 mmoVL glucose-induced insulin release significantly (P < .005 and P < .03. respectively). In order to examine the effects of FK 506 on the 50% maximal and slope of the insulin response to glucose, the data points from Fig 28 were normalized to the maximum response within each condition and were fitted using the Boltzmann equation: % = PO)/(I + exp) {P(2) x ([G)-P(3)} where [G)-glucose concentration, P(2) = glucose sensitivity. P(3) = 50% maximal. and P(l) is the maximal stimulation in either condition. FK 506. while inhibiting the maximal secretion induced by high glucose concentrations (Fig 28), produced a small apparent shift in the glucose dose-response curve to the left from a 50% maximum of 10.6 mmoVL in the control to 9.8 mmoVL in the FK 506-treated islets. At the 50% maximum, the slope of glucose sensing was increased. DISCUSSION FK 506 is a potent new immunosuppressant agent. When used long-term 1M in baboons. FK 506 had an apparent diabetogenic effect. 15 When the immunosuppression was changed at 4 days to an oral regimen. this was not observed in baboons undergoing renal transplantation 16 nor in cynomolgus monkeys undergoing pancreaticoduo-denal allotransplantation. 17 Preliminary studies in human 1.2 Ci: 0°·8 I.tJ N ~ :::!: a:: 00.4 z CARROLL, GONC;:ALVES, BOSCHERO ET AL • J:>. A • • control 6 cgE-~ 0.0 +-KKKKKKa---rlKKKKK::K-KKKKK---K---~---K----K ° 10 20 GLUCOSE (mM) Fig 3. Effect of FK 506 on the glucose dose-response slope and 50% maximum insulin secretion. To examine the elTects of 100 nglmL FK 506 on the slope of the glucose dose-response curve and the 50% maximum secretion, we fitted the data points from Fig 28 using the Boltzmann equation (see text). The data were noonalized to the maximum values in eadl condition. FK 506 produced a small shift in the 50% maximum along the x axis, the slope of glucose sensing was increased. recipients of liver allografts do not show a marked effect of this drug on carbohydrate tolerance when compared with historical controls who "ere treated "ith CyA_18 Thus far there have been few studies of the effect of this agent on islet cell function. FK 506 apparently does not alter morphology or interfere with insulin secretion from fetal islets of Langerhans. 19 Insulin secretion in neonatal and fetal islets is not comparable to the adult response,20.21 and since transplantation frequently involves tissue obtained from adult donors, we focused our studies on the effect of acute exposure to the drug on basal and glucose-stimulated insulin release. The present study shows that FK 506 does not inhibit basal insulin release. It does inhibit glucose-induced insu-lin release at the highest concentrations used 00 and 100 ng/mL). Our acute data are compatible with results that show a small inhibition after a 4-day culture of rat islets in the presence of FK 506. = These observations suggest that the inhibition is not cumulative at least during this time period. The amount of inhibition produced by FK 506 at high concentrations is similar to that produced by I and 10 #'&"mL CyA. Our results with CyA are comparable to those reponed by others.6 Of interest is the finding of an apparent shift in the glucose dose-response of insulin secretion in the presence of the drug. At lower glucose concentrations FK 506 does not inhibit insulin release and in fact some slight stimula-tion of secretion is observed. The inhibitioo of release observed at 16.7 and 22 mmoVL glucose represents a shift of the normal dose-response of the islets to the left (Fig 3). The mechanism of such a shift is not clear from the current studies. One possible explanation would be that FK 506 EFFECT OF FK 506 ON INSULIN RELEASE enhances calcium entry into the cells, which in high concentrations may be inhibitory in {3 cells as proposed by Draznin.23 These observations could have important clin-ical correlates. In summary, our studies and those of others indicate that inhibition of insulin release is apparent only at high concentrations of FK 506 and glucose. FK 506 appears to have considerable steroid sparing effect. II Thus, there may be fewer problems with the diabetogenic effect of this agent except when used IV, in very high concentrations, or when there are preexisting problems of carbohydrate me-tabolism. There are important species differences reported with side effects of this agent24 and further studies in humans are needed. Since there are potentially important applications of FK 506 in the field of diabetes, additional studies of effects of this agent on peripheral insulin action, glucose metabolism, response to non glucose secreta-gogues, effect on other islet hormones, and molecular mechanism of action on secretory cells and target cells are required. ACKNOWLEDGMENTS The authors acknowledge the technical assistance of Mr A. Heffess in some aspects of the work. Dr Venkatarmanan provided insights into the pharmacological propenies of the agents used. We acknowledge the assistance provided by the Laboratory of Statistical and Mathematical Methodology, NIH. We thank Dr E. Rojas for his many helpful discussions, review of the manuscript, and ongoing support. REFERENCES 1. White DTG: Cyclosporin A. New York. Elsevier Biomedi-cal, 1982 2. Yale JF, Grose M, Seemayer TA, et aI: Diabetes 36:749. 1987 339 3. Stiller CR, Dupre J, Gent M, et a1: Science 223:1362, 1984 4. Helmchen U. Schmidt WE, Siegel EG, et aI: Diabetologia 27:416. 1984 5. Andersson A. Borg H. Hallberg A. et aI: Diabetologia 27:66. 1984 6. Robertson RP: Diabetes 35: 1016. 1986 7. Chandrasekar B. Mukheljee SK: Biochem Pharmacol 37: 3609, 1988 8. NielsenJH, Mandrup-PoulsenT. SerupJ: Diabetes3S:I049, 1986 9. Robertson RP, Franklin G, Nelson L: Diabetes 38;S8, 1989 10. Kino T. Hatanaka H. Miyata S. et a1: Journal Antibiotic (Tokyo) 60:1256.1987 11. Starzi TE. Fung J, Venkataramman R. et aI: Lancet 2: 1000. 1989 12. Lacy PE, Kostianovsky M: Diabetes 16:3S, 1967 13. Scott AM. Atwater I, Rojas E: Diabetologia 21:470, 1981 14. Campbell G, Skillings JH: J Am Statistical Assoc 80:998, 1985 IS. Collier D St J, Caine R, Thiru S, et aI: Transplant Proc 29;397S, 1987 16. Todo S. Demetris A, Ueda y, et aI: Surgery 106:444, 1989 17. Ericzon BG. Kubota K, Groth CG, et aI: Transplant Proc 22:72. 1990 18. Mieles L, Todo S. Fung JJ. et a1: Transplant Proc 22;41. 1990 19. Leonard DK. Landry AS, Sollinger HW, et a1: Diabetes 38:172, 1989 20. Boschero AC. Tombaccini D, Atwater I: FEBS Lett 236: 37S, 1988 21. Rorsman p, Arkhammar p, BoItvist K, el aI: Proc Nat! Acad Sci USA 86;4S0S, 1989 22. Tze WJ, Tai J, Cheung S; Transplantation 49:1172, 1990 23. Draznin B: Am J Med 28:44, 1988 24. Thomson AW: Transplant Proc 22:100, 1990 

Effect of the immunosuppressant FK 506 on insulin release from adult rat islets of Langerhans.

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Effect of the immunosuppressant FK 506 on insulin release from adult rat islets of Langerhans.

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