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Acta Cirurgica Brasileira

Print version ISSN 0102-8650On-line version ISSN 1678-2674

Acta Cir. Bras. vol.12 no.3 São Paulo July/Aug./Sept. 1997

http://dx.doi.org/10.1590/S0102-86501997000300003 

3 – ARTIGO ORIGINAL

EXPERIMENTAL PANCREAS TRANSPLANTATION. THE CONSEQUENCES OF THE PORTOCAVAL SHUNT ON THE BLOOD GLUCOSE, PLASMA INSULIN, AND GLUCAGON.1

 

César Tadeu Spadella2
Maria Cecília Salgado Mercadante2
José Lúcio Martins Machado2
Silvana Artioli Schellini3
José Brandão Neto4

 

 

SPADELLA, CT.; MERCADANTE, M.C.S.; MACHADO, J.L.M.; SCHELLINI, S.A.; BANDÃO NETO, J. - Experimental pancreas transplantation: the consequences of the portocaval shunt on the blood glucose, plasma insulin, and glucagon.  Acta Cir. Bras.12(2):154-8, 1997.

SUMMARY: The consequences of the bypass of the portal venous effluent into the systemic circulation on the blood glucose, plasma insulin, and glucagon in immediate postoperative period of rats submitted to pancreas transplantation were studied. Forty outbred male Wistar rats were randomly assigned to two experimental groups: group NC included 20 non-diabetic control rats, submitted to simulated operating (sham-operated), and group PT included 20 diabetic rats that received heterotopic pancreas transplantation from normal donor Wistar rats. For 7 days prior and 1, 3, 6, 12, 24, 48, 72 and 96 hours after transplantation blood glucose, plasma insulin, and glucagon were recorded. These parameters were also concurrently recorded for NC rats. Diabetes was induced by i.v. alloxan administration; PT rats were immunosuppressed with cyclosporin A. NC rats presented normal values of blood glucose, plasma insulin, and glucagon over the course of experiment. A marked hyperinsulinemia was found in peripheral venous blood of PT rats, being plasma insulin significantly higher than that for NC rats (P<0.01) beginning 72- h after transplant. The plasma glucagon, elevated in pre-transplant period, did not change after transplant. Despite hyperinsulinemia and hyperglucagonemia, the blood glucose levels were elevated up to 6- h after transplant, but were within normal levels following this period. Beginning at 12- h after transplant the blood glucose levels observed in PT rats did not differ significantly to NC rats until the sacrifice.
SUBJECT HEADINGS: Portocaval shunt. Pancreas transplantation. Hyperinsulinism. Hyperglucagonemia.

 

 

INTRODUCTION

Although the portocaval shunt (PCS) has been described by Eck12 in 1877, the consequences of the bypass of the portal venous effluent directly into the systemic circulation are not still known.

Several physiological and pathological alterations have been reported after PCS2,9,11,21. However, some aspects of glucose metabolism and pancreatic hormones are controversial.

BEWICK et al.4 have related a state of hypoglycemia associated with a marked hyperinsulinemia in the first hours after heterotopic pancreatic transplantation in dogs that could led to death of the animals. This finding has been confirmed by others3,28, but also contested by several studies7, 20, 27.

In previous studies in our laboratory with pancreas transplantation and PCS in rats 25,31 we observed a number of deaths in the immediate postoperative period that the cause was not identified. It would be expected to ask whether the hyperinsulinemia with secondary hypoglycemia could not eventually explain them.

Therefore, the present experiment was performed in order to determine the metabolic profile of diabetic rats submitted to pancreas transplantation with systemic drainage. We hope to elucidate and to discuss the questions and controversies reported herein.

 

METHOD

Sample

Forty outbred male Wistar rats, weighing approximately 250g, were randomly assigned to two experimental groups: group NC consisted of 20 non-diabetic control rats submitted to simulated operating (sham-operated), and group PT consisted of 20 alloxan-diabetic rats that received heterotopic pancreas transplantation from normal donor Wistar rats.

Pancreas transplantation was performed according to the original procedure described by Lee et al.19, adapted to our laboratory30. Briefly, it consisted of a careful duodenopancreatectomy in the donor, with preparation of vascular trunks of the graft, which was revascularized in the recipient through the end-to side microsurgical anastomosis including a aorto-aortic anastomosis and a portocaval shunt.

Diabetes was induced by intravenous administration of alloxan* in a single dose of 42mg/kg body weight. Only diabetic rats with blood glucose levels above 200mg/dL, urine glucose levels above 3,000mg/dL, and severe clinical alterations as polydipsia, polyuria and polyphagia were included in the experiment. For prevention of rejection cyclosporin A** in a dose of 10mg/kg body weight per day was given by intraperitoneal via, beginning at 1st postoperative day.

Laboratory

Seven days prior and 1, 3, 6, 12, 24, 48, 72 and 96 hours after pancreas transplantation blood glucose, plasma insulin, and glucagon were recorded. These parameters were also concurrently recorded for NC rats. Blood samples for laboratory were obtained from the tail vein. For prevention of hypovolemia and hypotension a volume of 1-1,5ml of saline was injected into the penis vein after each collection. Blood glucose levels were determined by enzymatic method***insulin by radioimmunoassay in solid phase (coat-a-count insulin) and the glucagon by the double antibody technique****.

Statistical Analysis

Data were analysed according to Morrison’s profile analysis26, with level of significance set at 0.01 or 1%.

 

RESULTS

NC rats showed no evidence of clinical or laboratorial alterations throughout the experiment. All rats of this group remained healthy, within normal patterns of blood glucose, insulin, and glucagon levels. In contrast, all alloxan-diabetic rats presented in pre-transplant period high levels of blood glucose and glucagon associated with low values of plasma insulin. These values were significantly different (P<0.01) from NC rats (Table I).

 

 

Of the 20 PT rats, 3 rats died in the immediate postoperative period due to portocaval thrombosis (2 rats) and bleeding in the anastomosis sites (1 rat).

The glucose levels in peripheral venous blood of PT rats with technically successful transplants were still elevated at first 6- h after transplant. However, beginning at 12- h after transplantation, all rats of this group showed complete relief of hyperglycemia and complete restoration of plasma insulin levels, being significantly higher than that for NC rats beginning at 72- h after transplantation (P<0.01). The plasma glucagon, elevated in pre-transplant period, did not change within 96- h after transplant. Despite hyperinsulinemia and of hyperglucagonemia we did not observe hypoglycemia over the course of study.

The means of blood sugar observed for NC and PT rats throughout the follow-up are illustrated in Figure 1. The means of plasma insulin and glucagon are displayed in Figure 2.

 


Fig. 1 - Means of blood glucose in non-diabetic control rats (NC) and pancreas transplanted rats (PT) seven days prior (M0) and 1, 3, 6, 12, 24, 48, 72 and 96 hours of follow-up.

 

 


Fig. 2 - Means of plasma insulin and glucagon in non-diabetic control rats (NC) and pancreas transplanted rats (PT) seven days prior (M0) and 1, 3, 6, 12, 24, 48, 72 and 96 hours of follow-up.

 

DISCUSSION

Our results showed that rats submitted to intravenous administration of alloxan developed hyperglycemia, hypoinsulinemia, and hiperglucagonemia. These findings are primarily a consequence of the toxicity of alloxan on the beta-cells. Following necrosis of the islets, alloxan determines a decrease in secretion of insulin with a classical triphasic response of the blood glucose22 characterized by a initial hyperglycemia at 2 to 4 hours after the administration, a marked hypoglycemia at 6 to 12 hours, and a permanent hyperglycemia at 18 to 24 hours.

High plasma glucagon levels have also been considered a consequence of the damage on the beta-cells caused by alloxan. However, the mechanisms to explain the hyperglucagonemia after alloxan are controversial. GOMORI’s studies15 and posterior observations of MCLEAN and OGILVIE23 related a relative increase of the alfa and delta-cells after lesion of the beta-cells. Nevertheless, the results of these studies are discussed because there were no technical means in that time to distinguish the cellular components from the pancreas. HELLMAN and PETERSSON16 reported a slight increase of delta-cells population associated with a small decrease of the alpha-cells within 3 months after alloxan administration. This study was confirmed by Kobayashi et al.18, although, they have observed only a relative hyperplasia of the delta-cells.

MCEVOY and HEGRE24 reported a marked reduction of the beta-cells population within 7 days after alloxan administration. However, they did not observe significant alteration in the number of the alpha-cells, suggesting that the observed hyperglucagonemia after alloxan administration is not a consequence of increase of the alpha-cells. It is probably related to an increase in secretion by the normal number of the alpha-cells after the massive destruction of the beta-cells, being also possible that in the absence of the alpha-cell hyperplasia, an elevation in the nonpancreatic glucagon production in the intestines and salivary glands, might contribute to the hyperglucagonemia24.

GERICH et al.14 observed that long-term insulin infusion to insulin-deficient human diabetics corrected the hyperglucagonemia. Therefore, the hypoinsulinemia observed in alloxan-diabetic rats might also be an important cause of the high plasma glucagon levels. In our study, however, despite the supranormal insulin levels observed after pancreas transplantation, the elevated plasma glucagon in diabetic rats did not change over the course of experiment. These findings have been confirmed by others10,33.

The possible mechanisms of hyperglucagonemia and of the hyperinsulinemia observed after pancreas transplantation are also controversial. Idezuki et al.17 suggested that ischemia observed during graft manipulation leads to excessive insulin release from the graft during revascularization. This hypotesis has been confirmed by others6,8; however, ischemia only does not explain why plasma insulin remains within high levels for long-term after transplantation. Bergan et al.3 justified that hyperinsulinemia is only a consequence of the bypass of the portal venous effluent directly into the systemic circulation, bypassing the hepatic extraction mechanism.

The liver normally removes 40% of plasma insulin during the first passage of blood through the organ29. After pancreas transplantation, hepatic extraction of insulin does not occur, resulting in hyperinsulinemia; the same probably happens with glucagon. However, despite hyperinsulinemia and hyperglucagonemia, in our experiment the blood glucose levels were within normal values after pancreas transplantation. These findings have been reported by several studies1,7,10,17,32.

Why blood glucose remains within normal levels despite hyperinsulinemia is another good question. Froesch et al.13 reported that the liver converts inactive atypical insulin, produced by the pancreas, into active typical insulin. It is possible, then, that a large percentage of plasma insulin may be of the inactive atypical type, explaining hyperinsulinemia with normoglycemia. It is also possible that graft denervation causes an increase in the release of inactive insulin rather than active insulin5. However, neither denervation of the pancreas alone nor bypassing the hepatic circulation alone can reproduce these results. It appears that hyperinsulinemia and hyperglucagonemia following heterotopic pancreatic transplantation are the result of a combination of the two factors: denervation causing maximal insulin release from the pancreas and blocking the inhibitory function of glucose on the insulin and glucagon levels, and total venous transposition impeding complete hepatic extraction of the hormones from the blood.

These theories, however, are not generally accepted because hypoglycemia and hyperinsulinemia have been observed in studies on heterotopic pancreatic transplantation. BEWICK et al.4 found hypoglycemia and hypokalemia within 2 to 3 hours after pancreatic allotransplantation in dogs, which could led to death of the animals whether were not corrected by parenteral dextrose and potassium or prevented by parenteral cortisone given at the time of transplantation. These results were subsequently reported by other workers3,28.

In our study as seen we did not observe neither hypoglycemia nor deaths despite hyperinsulinemia. It is possible that other factors as prolonged fasting, hypovolemia, hypotension etc., not observed in our study, may be participating in the genesis of those alterations. We believe, however, that the exact mechanism of hyperinsulinemia and of hyperglucagonemia, with its consequences on the blood glucose, still remains to be elucidated.

 

CONCLUSION

Rats submitted to i.v. administration of alloxan presented hyperglycemia, hypoinsulinemia and hyperglucagonemia. These alterations were corrected by pancreas transplantation, except hyperglucagonemia.

Plasma insulin was significantly elevated beginning at 72- h after pancreas transplantation. Despite hyperinsulinemia and hyperglucagonemia, the blood glucose levels were within normal levels over the course of experiment beginning at 12- h after transplantation. Hypoglycemia leading to death was not observed in the first hours after heterotopic pancreas transplantation. It is possible that other mechanisms, not addressing in this study, may explain these complications.

Acknowledgments

We thank Prof. Paulo R. Cury for statistical analysis, Mrs. Irene Spago for skilled laboratorial assistance and Ms. Silvana A. Cappelletti and Licemara M. Montagna for typed of the manuscript.

 

REFERENCES

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4. BEWICK, M.; MUNDY, A.R.; EATON, B.; WATSON, F. - Endocrine function of the heterotopic pancreatic allotransplant in dogs. II. Immediate post-transplant period. Transplantation, 31: 19-22, 1981.        [ Links ]

5. BEWICK,M.; MUNDY, A.R.; EATON, B.; WATSON, F. - Endocrine function of the heterotopic pancreatic allotransplant in dogs. III. The cause of hyperinsulinemia. Transplantation, 31: 23-5, 1981.        [ Links ]

6. BINDER, P.S.; HEISKELL, C.A.; ADAMS, J.R.; CERRA, F.B.; EILERT, J.B.; BERGAN, J.J. - Pancreatic function after normothermic ischemia. I. In vivo ischemia. Amer. J. Surg., 120: 687-92, 1970.        [ Links ]

7. BREKKE, I.B.; GULLESEN, I.; REFSUM, S.B.; FLATMARK, A. Long-tem endocrine function of duct-ligated pancreas isotransplants in rats. Europ. Surg. Res., 12: 167-78, 1980.        [ Links ]

8. CERRA, F.B.; ADAMS, J.R.; EGGERT, D.E.; EILERT, J.B.; BERGAN, J.J. - Pancreatic function after normothermic ischemia. II. Cadaveric transplantation. Amer. J. Surg., 120: 693-6, 1970.        [ Links ]

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11. DUY, N.; YAMAGUCHI, Y.; PRABHUDESAI, M.; BABB, J.; GANS, H. - Cancer of the bladder in the portocaval shunted rat. Gastroenterology, 80: 1331, 1981.        [ Links ]

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13. FROESCH, E.R.; JAKOB, A.; ZAHRID, G.R.; SIMON, E. - Immunoreactive insulin, suppressible, and non- suppressible insulin-like activies in rats after administration of glucose and induction of hyperglycaemia by mannoheptulose. Diabetologia, 2: 265-8, 1966.        [ Links ]

14. GERICH, J.E.; TSALIKIAN, E.; LORENZI, M.; SCHNEIDER, M.; BOHANNON, V.; GUSTAFSON, G.; KARAM, J.H. - Normalization of fasting hyperglucagonemia and excessive glucagon responses to intravenous argine in human diabetes mellitus by prolonged infusion of insulin. J. Clin. Endocrinol. Metab., 41: 1178-80, 1975.        [ Links ]

15. GOMORI, G. - Observations with differential stains on human islets of Langerhans. Amer. J. Path., 17: 395-406, 1941.        [ Links ]

16. HELLMAN, B. & PETERSSON, B. - Long-term changes in the alpha 1 and alpha 2 cells in the islets of Langerhans of rats with alloxan diabetes. Endocrinology, 72: 238-42, 1963.        [ Links ]

17. IDEZUKI, Y.; FEEMSTER, J.A.; DIETZMAN, R.H.; LILLEHEI, R.C. - Experimental pancreaticoduodenal preservation and transplantation. Surg. Gynec. Obstet., 126: 1002-14, 1968.        [ Links ]

18. KOBAYASHI, K.; TAKAHASHI, Y.; JOHSHITA, T. - Influence of alloxan administration and hypophysectomy on the pancreatic islets of the rat. Arch. Histol. Jap., 25: 199-216, 1964.        [ Links ]

19. LEE, S.; TUNG, K.S.K.; KOOPMANS, H.; CHANDLER, J.G.; ORLOFF, M.J. - Pancreatiacoduodenal transplantation in the rat. Transplantation, 13: 421-5, 1972.        [ Links ]

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21. LINNEMANN, U.; KUCH, P.; SCHWILLE, O. P. - Ammonium urate urolithiasis in the rat with portocaval shunt: some aspects of mineral metabolism and urine composition. Urol. Res., 14: 319-22, 1986.        [ Links ]

22. LUKENS, F.D.W. - Alloxan diabetes. Physiol. Rev., 28: 304-30, 1948.        [ Links ]

23. MacLEAN, N. & OGILVIE, R.F. - Quantitative estimation of the pancreatic islets tissue in diabetic subjects. Diabetes, 4: 367-76, 1955.        [ Links ]

24. McEVOY, R.C. & HEGRE, O.D. - Morphometric quantitation of the pancreatic insulin-, glucagon-, and somatostatin- positive cell populations in normal and alloxan-diabetic rats. Diabetes, 26: 1140-6, 1977.        [ Links ]

25. MERCADANTE. M.C.S.; MACEDO, A.R.; RODRIGUES, M.A.M.; DAYOUB, M.C.O. - Pre-neoplastic lesions in the urinary bladder of rats with portocaval shunt. Acta Cir. Bras., 11: 27-31, 1996.        [ Links ]

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27. ORLOFF, M.J.; LEE, S.; CHARTERS, A.C.; GRAMBORT, D.E.; STORCK, G., KNOX, D. - Long-term studies of pancreas transplantation in experimental diabetes mellitus. Ann. Surg., 182: 198-206, 1975.        [ Links ]

28. RUIZ, J.O.; UCHIDA, J.; SCHULTZ, L.S.; LILLEHEI, R.C. - Function studies after auto- and allotransplantation and denervation of pancreaticoduodenal segments in dogs. Amer. J. Surg., 123: 236-42, 1972.        [ Links ]

29. SAMOLS, E. & RYDER, J.A. - Studies on tissue uptake of insulin in man using differential immunoassay for endogenous and exogenous insulin. J. Clin. Invest., 40: 2092-102, 1961.        [ Links ]

30. SPADELLA, C.T. - Avaliação funcional do transplante pancreato-duodenal no rato diabético. Estudo clínico, laboratorial, anatomopatológico e imunohistoquímico. Botucatu, 1989. 221p. (Tese - Doutoramento - Faculdade de Medicina, Universidade Estadual Paulista).        [ Links ]

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32. UEDA, T.; LEE, S.; STORCK, G.; ORLOFF, M.J. - Metabolic effect of alloxan diabetic rats before and after pancreas transplantation. Proc. Anat. Microsurg. Soc., 3: 423-4, 1979.        [ Links ]

33. VAN HOORN, W.A.; VINIK, A.J.; VAN HOORN-HICKMAN, R. - A comparison of two methods of orthotopic pancreas transplantation in the pig. Brit. J. Med., 26: 287-91, 1978.        [ Links ]

 

 

SPADELLA, C.T.; MERCADANTE, M.C.S.; MACHADO, J.L.M.; SHELINI, S.A.; BRANDÃO NETO J. - Transplante experimental de pâncreas: as consequências da derivação portocava na glicemia sanguínea, na insulina plásmica e no glucagon. Acta Cir. Bras., 12(3):154-8, 1997.

RESUMO: A proposição foi realizada, no período pós-operatório imediato, em 40 ratos Wistar, distribuídos por sorteio em doi grupos: grupo NC, vinte ratos correspondentes ao grupo controle, não diabético, submetidos a operação simulada e o grupo PT, 20 ratos correspondentes ao grupo diabético que recebeu transplante de pâncreas heterotópico de ratos Wistar normais. Durante sete dias, antes do transplante, e 1, 3, 6, 12, 24, 48, 72, 96 horas após, determinava-se a glicose sanguínea, a insulina plasnática e o glucagon. Estes parâmetros eram obtidos também do grupo NC. Diabetes mellitus experimental era induzida pela administração intravenosa de aloxana. O grupo PT era imunosuprimido com ciclosporina A. O grupo NC apresentou níveis normais de glicose sanguínea, de insulina plásmica e de glucagon, durante todo o experimento. Foi encontrada nítida hiperinsulinemia no sangue venoso periférico do grupo PT. A insulina plasmática era significantemente maior no grupo PT comparada ao grupo NC começando 72 horas após o transplante. O glucagon plasmático, elevado no período pré-transplante, não se alterou após o transplante. Apesar de hiperinsulinemia e hiperglucagonemia, os níveis de glicose sanguínea eram elevados 6 horas após o transplante e mantiveram-se normais após este período. Considerando-se os níveis de glicose sanguínea 12 horas pós-transplante, não houve diferença estatisticamente significante entre os grupos PT e NC, até o sacrifício.
DESCRITORES: Derivação portocava cirúrgica. Transplante de pâncreas. Hiperinsulinismo. Hiperglucagonemia.

 

 

Address reprint request to:
César Tadeu Spadella, MD, PhD
Departamento de Cirurgia e Ortopedia
Faculdade de Medicina - UNESP
18618-970 Botucatu - São Paulo - SP

Accepted for publication on march, 1997.

 

1. Paper from the Laboratory of Surgical Technique and Experimental Surgery of Faculdade de Medicina de Botucatu, Universidade Estadual Paulista (UNESP).
2. Department of Surgery, Faculdade de Medicina de Botucatu, UNESP.
3. Department of Ophthalmology, Faculdade de Medicina de Botucatú, UNESP.
4. Department of Clinica Médica, Faculdade de Medicina de Botucatu, UNESP. Research Supported by FAPESP, FUNDUNESP and CNPq
* J.T. Baker Chemical Co., Phillipsburg, New Jersey
** Sandoz, Basle, Switzerland
*** CELM Equi. Lab., São Paulo, Brazil
**** Diagnostic Products Co., Los Angeles, CA

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