Exenatide (exendin-4) can reduce blood glucose levels, increase insulin secretion and improve insulin sensitivity through mechanisms that are not completely understood.

In the present study, we examined the effects of exenatide treatment on glucose tolerance (intravenous glucose tolerance test), insulin sensitivity (euglycaemic-hyperinsulinaemic clamps), insulin signalling (insulin receptor substrate 1 tyrosine phosphorylation) and adipocytokine levels (visfatin and adiponectin) in high fat-fed rats.

Administration of exenatide (0.5 or 2.0 mug/kg twice daily x 6 weeks) prevented high-fat diet (HFD)-induced increases in body weight, plasma free fatty acids, triglycerides and total cholesterol. Exenatide also prevented HFD-induced deterioration in peripheral and hepatic insulin sensitivity, insulin clearance, glucose tolerance and decreased tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) in fat and skeletal muscles. Interestingly, plasma visfatin levels decreased in exenatide-treated rats, whereas expression and plasma levels of adiponectin increased.

These results indicate that chronic exenatide treatment enhances insulin sensitivity and protects against high fat-induced insulin resistance.

Insulin resistance and insulin deficiency are proposed as risk factors for IGT and type 2 diabetes. We assessed the predictive value of initial parameters for the outcome of an OGTT performed 24.3+/-2.9 years later in an unselected healthy non-obese population.

The K-value of an IVGTT was determined in 267 healthy subjects (mean+/-SD: age 31.0+/-12.0 years, BMI 21.8+/-2.8 kg/m(2)). First-phase insulin response to a glucose infusion test was estimated as an incremental 5- or 10-min (DeltaI5 or DeltaI10) value, and as insulinogenic indices (DeltaI5/DeltaG5 or DeltaI10/DeltaG10) adjusted for insulin sensitivity determined by homeostasis model assessment for insulin resistance ([DeltaI5/DeltaG5]/HOMA-IR).

At follow-up, six subjects had type 2 diabetes and 47 had IGT; 214 retained normal glucose tolerance. Insulin sensitivity and early (30 min) insulin response decreased with decreasing outcome OGTT. Blood glucose (2 h) at OGTT correlated positively with initial age and BMI, and negatively with DeltaI5/DeltaG5, (DeltaI5/DeltaG5)/HOMA-IR and K-value. In multiple linear regression analysis, (DeltaI5/DeltaG5)/HOMA-IR, DeltaI10, K-value, age, HOMA estimate of insulin secretion, and fasting plasma glucose were significantly associated with 2-h OGTT blood glucose. Similar results were obtained on comparing differences between subjects with normal and decreased (IGT+diabetes) glucose tolerance.

In 267 non-obese healthy subjects, initial K-value and first-phase insulin response to glucose adjusted for insulin sensitivity, but not insulin sensitivity itself, were strong predictors of the outcome of an OGTT performed 25 years later. Thus, in contrast to obese or other high-risk populations, in lean subjects, decreased beta cell function, but not insulin resistance itself, determines future glucose tolerance.

Although an attractive alternative to daily insulin therapy, allogeneic pancreatic islet transplantation has yielded suboptimal results in clinical trials, in contrast to islet allotransplantation in animal models, which have demonstrated consistent success. The successful transplantation of isolated islets to the thymus, with a single concomitant dose of antilymphocyte serum, has been demonstrated in rodents, and more significantly, such intrathymic islet allografts have been shown to induce recipient tolerance toward subsequent extrathymic donor strain islet allografts. Intrathymic islet autotransplantation has been pursued, as a prelude to studies of allogeneic IT islet transplantation and tolerance induction, in canine, porcine, and non-human primate models, to assess the large animal thymus as a site capable of supporting a viable islet graft. However, little functional or histological evidence has established definitive survival of islets transplanted within the thymus of a phylogenetically advanced species, which may be requisite to tolerance induction. This study describes the successful intrathymic autotransplantation of isolated islets using a canine model.

Purpose-bred juvenile dogs, aged 4-6 months, underwent partial (n=4), or total pancreatectomy (n=11), and transplantation of autologous islets. The pancreas (or pancreatic limb) was distended with collagenase solution, and digested using a modification of the semiautomated system of Ricordi. Islets were purified by discontinuous gradient centrifugation, using Euroficoll (ficoll in Euro-Collin's kidney preservation solution). Partially pancreatectomized canines underwent IT transplantation of purified autologous islets (8000+/-4000 IEs), and were killed 8 weeks posttransplant. Totally pancreatectomized canines underwent transplantation of autologous islets to the liver (via portal vein embolization, n=5, IPO group) or the thymus (via direct IT injection, n=6, IT group), and were serially evaluated for a period of 8 weeks posttransplant to assess fasting blood glucose (FBG), serum insulin (SI) levels, and i.v. glucose tolerance (IVGTTs). K values (defined as the %-decrease/minute of the log(e) of blood glucose values) were calculated from IVGTT results.

After autotransplantation in this cohort of animals, five of five IPO, and three of six IT islet recipients, remained normoglycemic (mean FBG< or =250 mg%) immediately posttransplant, and all recipients exhibited significantly elevated SI levels compared to apancreatic controls (n=10, followed 72 hr postpancreatectomy). Normal k values (=-1.1) were observed in two of five IPO, and in one of six IT recipients, 8 weeks after transplantation, and thymic tissue insulin content was increased compared to non-islet-bearing thymi (93.7+/-48.6 ng/g tissue vs. 0.7+/-0.4 ng/g tissue). At 8 weeks posttransplantation thymi from both partially and totally pancreatectomized animals were resected and processed for histological examination. Microscopic analysis of islet-bearing thymi revealed positive staining for islet-specific hormones (insulin and glucagon) within all IT recipients., Identification of islets within thymi of hyperglycemic IT recipients was problematic as islet beta cells were highly degranulated as a result of the recipients glycemic state.

These results indicate that autologous islets, transplanted to the canine thymus, engraft, function, and survive for up to 8 weeks after islet autotransplantation to the canine thymus and establish the feasibility of intrathymic islet transplantation in a phylogenetically advanced animal model. The ability of islets to survive within the thymic environment for a period of at least 8 weeks after transplantation suggests that the successful induction of specific unresponsiveness secondary to intrathymic transplantation will not be impaired or limited by the inability of a viable islet mass to survive within the thymus for a sufficient period.

The inability to diagnose early rejection of an islet allograft has previously proved to be a major impediment to progress in clinical islet transplantation. The need to detect early rejection will become even more relevant as new tolerance-inducing protocols are evaluated in the clinic. We explored three novel approaches toward development of early diagnostic markers of islet rejection after islet allotransplantation.

(a) Canine islet allograft transplant recipients were immunosuppressed for 1 month, then therapy was withdrawn. Serum glutamic acid decarboxylase antigen (GAD65), an endogenous islet protein, was monitored daily with a CO2 release assay. (b) Rodent islets were genetically engineered to express a unique foreign protein (beta-galactosidase) by using adenoviral vectors, and after allograft transplantation, the viral-specific protein was measured in serum using optical luminescence. (c) Rodents receiving islet allografts were immunosuppressed temporarily, and daily glucose tolerance tests were followed until graft failure occurred.

(a) Although serum monitoring of GAD65 antigen demonstrated elevated levels preceding loss of graft function in preliminary studies, the effect was not reproducible in all animals. (b) Genetically engineered rodent islets demonstrated normal insulin kinetics in vitro (insulin stimulation index 2.57+/-0.2 vs. 2.95+/-0.3 for control islets, P=ns), and purified viral protein products had a stable half-life of 8 hr in vivo. After islet allotransplantation, there were two peak elevations in serum viral proteins, confirming that an intra-islet "sentinel signal" could be detected serologically during acute rejection. There was no lead-time ahead of hyperglycemia, however. (c) Daily sequential intravenous glucose tolerance (IVGT) tests demonstrated evidence of allograft dysfunction (decline in KG) with a 2-day lead time to hyperglycemia (2.58+/-0.3 vs. 1.63+/-0.2%/min, respectively, P<0.001), with an accuracy of 89%, sensitivity of 78%, and specificity of 95%.

Of the three diagnostic tests, metabolic assessment with an abbreviated IVGT was the most effective method of demonstrating early islet dysfunction due to rejection.

In islet transplantation pancreatic preservation before islet isolation is an obstacle compromising islet yield and viability. We tested the feasibility of a two-layer method (University of Wisconsin solution [UW]/perfluorochemical) for pancreatic preservation before islet isolation.

Dog pancreases were processed into pure islets by the method of Ricordi preceded by five different preservations (groups 1-a and 1-b, the two-layer method for 3 and 24 hours; groups 2-a and 2-b, simple cold storage in UW for 3 and 24 hours; group 3, without preservation). Islet yields and functional success after autotransplantation into the liver were compared among the groups.

Postpurification islet equivalents (IE)/gm pancreas and functional success rate were 5600 (mean), 83% in group 1-a; 4000, 56% in group 1-b; 4700, 33% in group 2-a; 1300, 0% in group 2-b; and 5000, 89% in group 3 (p < 0.05; 2b versus 1-a, 1-b, and 3), respectively. There was no statistical difference among groups 1-a, 1-b, and 3 in terms of islet yield and function (p > 0.2).

The two-layer method is more effective than conventional simple cold storage in UW for pancreatic preservation before islet isolation. Clinical trials with the two-layer method are warranted.

It is not known whether discordant, free, nonvascularized xenoislets-akin to discordant, vascularized, solid xenoorgans-are hyperacutely rejected. Quantitative xenoislet requirements and the optimal transplant site also remain to be defined.

We studied these questions with a discordant dog-to-diabetic Lewis rat xenoislet model, using (1) functional (cure of streptozotocin-induced diabetes) and (2) histologic (biopsies of intraportal grafts) parameters. WF-to-Lewis alloislet recipients served as histologic controls.

(1) We found that 5000 xenoislet equivalents (IEs) transplanted into the portal veins of nonimmunosuppressed rats never functioned. Peritransplant combination therapy (rapamycin, cyclosporin A, anti-rat lymphocyte serum) significantly prolonged graft survival of 5000 intraportal IEs (median, 3 days) but not of 2500 intraportal or of 5000 intraperitoneal or renal subcapsular IEs. (2) By means of immunofluorescence (at 1 hour after transplantation), we noted immunoglobulin M (IgM) and IgG binding to islets in xenografts but not allografts; we noted complement and fibrinogen binding in both xenografts and allografts. Insulin-positive islet cells within intact xenoislets were demonstrated in nonimmunosuppressed rats up to 48 hours after transplantation. Cellular xenograft infiltration and inflammation, beginning at 6 hours, were observed even in immunosuppressed rats. (3) Thus, in spite of IgM and IgG binding, intraportal discordant xenoislets were not hyperacutely rejected and destroyed. Nevertheless, universal xenoislet nonfunction in nonimmunosuppressed rats was immune mediated. A large xenoislet mass (more than 10,000 IEs/kg), the intraportal site, and combination therapy were absolute prerequisites for immediate function. But even if the prerequisites were all fulfilled, accelerated xenoislet graft failure occurred.

This outcome suggests that the specific binding of IgM and IgG to xenoislets, in conjunction with the binding of complement and fibrinogen, contributed to accelerated graft failure. Thus distinction between discordant and concordant species combinations is important for free, nonvascularized xenoislet transplants. These findings and the steroid-free combination protocol (rapamycin, cyclosporin A, anti-T-cell therapy) warrant further testing in preclinical discordant xenoislet studies.

Intraductal distention of the pancreas with collagenase followed by stationary warm incubation improves the recovery of islets of Langerhans in the rat, but controlled studies are needed for valid comparison with standard isolation methods. We have modified Gotoh's technique of stationary digestion for high-yield isolation in the rat (Stationary). The method is subjected herein to rigorous blinded comparison with the standard chopped tissue (Chopped) technique, based on Lacy et al., as performed in our laboratory for over 10 yr. Islet recovery was determined by a single observe 'blinded' to the method of isolation used, and only intact islets of diameter > or = 100 microns were included. Stationary gave 719 +/- 114 islets per pancreas (mean +/- SD, n = 21 isolations) vs. 487.5 +/- 69 for Chopped (n = 36 isolations), a 47.5% increment in yield (p < 0.0001). In vitro islet perifusion showed no statistical difference in stimulation index (SI) or stimulated area under the curve (SAUC) between the two methods, but Stationary showed a trend towards improved phase II insulin release. In vivo function was assessed by isogeneic transplantation of 2,000 islets beneath the renal capsule of streptozotocin diabetic recipients (65 mg/kg Sigma); Stationary recipients (n = 7) became normoglycemic (< or = 8 mmol/L) by 3.3 +/- 4.8 days vs. 1.6 +/- 1.5 days for Chopped recipients (p = 0.4 ns, mean +/- SEM). IVGTT performed at 1 mo posttransplant gave K-values for Stationary of 2.64 +/- 0.8 vs. 2.62 +/- 0.8 for Chopped (mean +/- SD, p = 0.9 ns, n = 6, unpaired t-test), which were not distinguishable from normal control rats (2.59 +/- 0.8) (p = 0.9 ns, n = 10). Graft function remained stable until graft bearing nephrectomy induced hyperglycemia uniformly within 1 day. Graft histology showed a healthy well-preserved structure on light microscopy, with well-granulated beta cells on EM. Economic costs of rat, collagenase, and Ficoll were 26% ($50.82) lower per recipient for Stationary. We conclude that modified stationary digestion significantly improves islet recovery with excellent in vitro and in vivo function, and is cost effective.

A copper deficient diet is reported to reduce acinar tissue in vivo. We investigated the suitability of this method to reduce in vivo acinar tissue mass of a rat pancreas prior to transplantation of dispersed pancreatic tissue. We also studied islet function in the acinar depleted pancreas and the outcome of transplantation of islets from such pancreata. Eighty-two Wistar Furth rats were divided into two groups with 42 animals in the control group receiving regular diet, and 40 receiving copper deficient diets (Cudt) plus tetraethylene- pentamine penta-hydrochloride (TEPA) as a chelating agent. All animals in the control group and 34 (85%) in the Cudt group tolerated this diet and survived for 60 days or longer. At the end of 60 days, all experimental animals were converted to a regular diet until the pancreata were harvested for islet transplantation. Eight rats in the Cudt group, which were converted to a regular diet for 2 weeks, and 2 in the control group were randomly selected and sacrificed to study the pancreas for acinar depletion and islet morphology. An intravenous glucose tolerance test (IVGTT) in the control group (n=24) and the Cudt group (n=25) showed K-values of 1.891+/-0.7 and 1.107+/-0.47, respectively (P-ns). Histology of pancreata showed normal acinar tissue in the control group and reduction of acinar tissue mass in the Cudt group. Furthermore, immunohistochemistry for insulin, glucagon, and somatostatin showed positively staining, while amylase was negative in the Cudt group, compared with the positive stain for cells in the control group. Standard collagenase digestion of the pancreas showed islets were surrounded by scant amounts of acinar tissue in the Cudt group compared with the control group. The islet count in the control group was 523+/-126 and 611+/-52 in the Cudt group. The mean volumes of dispersed pancreatic tissue were 0.3875+/-0.14 and 0.0668+/-0.029 ml per rat in the control and Cudt groups, respectively (P<0.05). Transplantation of dispersed pancreatic tissue from the control group into the spleen of two diabetic Wistar Furth rats resulted in the death of the recipients within 24 hr. To avoid this complication, purified islets from the control group were used for transplantation. Purified islets from 5 donor pancreata from the control group and dispersed pancreatic tissue from 3 pancreata in the Cudt group were transplanted into each recipient. Islet function was seen in 75% of the rats transplanted with purified islets from the control group, and in 67% receiving dispersed pancreatic tissue from the Cudt group. Rats with sustained islet function for 30 days following islet transplantation developed diabetes following splenectomy. The islet cells were positively stained for insulin these splenectomy specimens. This study demonstrates that rats maintained on a copper deficient diet for 60 days show depletion of collagenase digested volume of whole pancreatic tissue occurred in the Cudt as compared with the control group. Transplantation of dispersed pancreatic tissue from the acinar depleted pancreas was successful in reversing diabetes. We conclude that Cudt containing TEPA depletes exocrine tissue and facilitates pancreas digestion for successful transplantation of islets into the portal system.

Post-ischemic injury is one of the most important problems affecting successful organ procurement and transplantation. The present study was performed to determine whether alpha-tocopherol can protect the endocrine function of pancreatic grafts against ischemia-reperfusion injury during rat heterotopic pancreatic transplantation. Rats with streptozotocin-induced hyperglycemia were used as recipients. The donor pancreas was removed and subjected to warm ischemia at 37 degrees C for 30, 60, 90 and 120 min, and then transplanted into a recipient. A 30-min period of warm ischemia did not impair the endocrine function of the pancreatic grafts, which was assessed by measuring the blood glucose levels and glucose decay constants (K), and a 60-min period of warm ischemia was considered to be the critical period for reversible tissue damage. Pretreatment with alpha-tocopherol (20 mg/kg/day, i.v.) for seven days before graftectomy significantly decreased blood glucose levels to less than 200 mg/dl and significantly increased K values in the recipient rats after transplantation when compared with placebo pretreatment. These results suggest that alpha-tocopherol pretreatment can protect the endocrine function of pancreatic grafts against injury due to warm ischemia followed by reperfusion.

The objective of this study was to analyze the impact of rapamycin and/or cyclosporine on the metabolic efficiency of intrasplenic islet autografts in dogs. An insulin modified frequently sampled intravenous glucose tolerance test was carried out before, on the last of 30 days treatment with drug and 30 days after cessation of drug therapy in dogs with stable function 1 to 7 years after total pancreatectomy and intrasplenic islet autografting. Analyses were performed for glucose clearance, insulin release, insulin sensitivity, and other variables. Rapamycin treatment was associated with increased glucose clearance, increased total and stimulated insulin release in response to glucose, and increased fasting plasma insulin level, as well as reduced insulin clearance. Cyclosporine at 300 micrograms/L had little impact on the measured variables. Treatment with rapamycin and cyclosporine showed a similar (although less-marked) pattern of changes to rapamycin alone. Rapamycin, with or without concomitant cyclosporine, was not associated with adverse impact on islet function or glucose metabolism in this canine model of pancreatic islet transplantation.

Previous studies of intraportal islet autotransplantation in large animals have reported graft failure after months or years. In the rat it has been reported that intraportal islet isografts eventually failed whilst islets transplanted to the renal subcapsule functioned up to a year. We made Dark Agouti (DA) rats severely diabetic with streptozotocin, then 1000 or 3000 DA islets were transplanted beneath the renal capsule or into the liver. One set of transplanted rats and untreated diabetic and normal non-diabetic littermates were monitored lifelong by measurement of plasma glucose, others were killed at 6, 12 and 18 months for measurement of haemoglobin A1c, intravenous glucose tolerance test, pancreas insulin content and histology of the kidney. Renal glomerular basement membrane thickness was measured by the orthogonal intercept method. The results showed that intraportal isografts reversed hyperglycaemia significantly faster than renal subcapsular isografts. In the renal subcapsular site, consistent reversal of diabetes was achieved with 3000 islets but not with 1000 islets. Furthermore, intraportal islet grafts with 3000 islets led to lower, normal random glucose level than renal subcapsular grafts for the first 13 months. Normoglycaemia was maintained lifelong in all rats that achieved early normoglycaemia after transplantation of 3000 islets, irrespective of the site of islet transplantation. The fasting glucose, haemoglobin A1c levels, K value and glomerular basement membrane thickness of the recipients of 3000 islets to either the intraportal and subcapsular site were not significantly different from each other and the normal controls up to 18 months.(ABSTRACT TRUNCATED AT 250 WORDS)

In a series of experiments on syngeneic rat islet (pancreatic fragments) transplantation we demonstrate that direct hepatic transplantation is successful to alleviate streptozotocin induced diabetes with tissue from a single donor. The experimental groups were: recipients of fresh, and cryopreserved pancreatic fragments. The fresh graft was prepared by collagenase digestion. Cryopreserved fragments were further treated by a standard freeze-thaw protocol which consists of slow cooling at 0.3 degrees C/min to -75 degrees C followed by transfer to -196 degrees C, in the presence of 1.4 M Me2SO, and storage at this temperature for one day or 1 wk, and then warming them back to room temperature at a rate of 35 degrees C/min. Streptozotocin-induced diabetes in rats can be reversed by injection of isolated pancreatic fragments from a single donor directly into the liver. No significant difference was observed between the recipients receiving fresh or cryopreserved tissue for 1 day or 1 wk. It is possible that elaborate purification itself is not conducive to successful alleviation of diabetes. This would corroborate the hypothesis that trophic factors are present in impure fragments. The direct infusion of islet fragments into the liver could allow for percutaneous administration in human transplantation.

Two models of pancreas transplantation were examined in the rat with a view to choosing one for regular use in functional experiments. A cuff technique applied to the renal vessels of the recipient was used. The histology and endocrine functioning of whole pancreas-duodenal transplant (Tx) plus bladder drainage (drainage model), and the pancreatic duct ligated segmental pancreas Tx (ligated model) were examined. Syngeneic operations were performed using either Lewis or Wistar rats. Streptozotocin (STZ) 60 mg/kg intravenously (i.v.) was used to render the recipient rats diabetic. A cuff technique was used with both models to anastomose the grafts to the renal vessels instead of the conventional technique of hand suturing to the abdominal vessels. This allows a shorter warm ischaemia time for the donor pancreas and leaves the systemic circulation intact leading to a high success rate for both techniques. Operation survival rates were 93% (n = 30) and 90% (n = 10) in the ligated and drainage models, respectively. The recipients in both groups were normoglycaemic for > 100 days. Histological examination revealed atrophic exocrine tissue early in the ligated group but only two from the drainage group showed exocrine atrophy at > 100 days. There was no statistical difference in i.v. glucose tolerance tests with both models showing a normal pattern. Thus, endocrine function remained independent of exocrine function. Both models are quicker than the conventional techniques. The duct ligation model was a simpler transplant to perform, suggesting that this should be the technique of choice in future experiments.

As far as we know, after adult enzyme-digested pancreatic fragment autotransplantation, the fate of the inoculated pancreatic tissue has never been reported and the hypothetic engrafted islet mass growth by mitotic division or by a true islet neogenesis from ductular precursor cells has never been demonstrated. In dogs with total or near-total (90%) pancreatectomy that preserves the duodenum and the common bile duct, morphologic study of the pancreatic tissue inoculated into the spleen has demonstrated an exuberant ductular-acinar-islet regenerative process, with progressive cystic degeneration of the newly formed ductular-acinar structures occurring simultaneously with the selective survival and growing predominance of extraductal tissue scattered as distinct islets, clusters of islet cells, or single islet cells. In addition to the B, A2, and D cell types of the normal adult dog islet, we have also seen a peculiar ultrastructural pleomorphism of the insular B cells, frequently combined with their ductular or glandular arrangement in maturing islets. Rare or never before reported islet cell types in the adult dog's islets (G cells, mixed endocrine cells of the A2-D, D-B, and A2-B types, and mixed acinar-islet cells of the D-acinar type) were also putatively identified. Using light microscopy we have identified many mitotic figures on ductular and centroacinar cells in ductules and ductular-acinar structures.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic pancreatitis (CP) leads to deterioration of the endocrine pancreatic function by fibrotic destruction. The aim of the present study was to investigate whether resection or duct drainage in patients with CP would have a direct impact on the pancreatic beta cell function. An intravenous glucose tolerance test (IVGTT) was performed before, after and in some cases 3 months after operation in ten patients each of whom had been treated by either resection or duct drainage. Three patients undergoing pancreatic resection for cancer served as controls. Beta cell function was assessed by glucose elimination (K-values), insulin and C-peptide response. K-Values in patients with CP were not significantly influenced after resection (1.93 +/- 0.78/2.13 +/- 0.72; n.s.) or drainage (1.26 +/- 0.47/1.54 +/- 0.58; n.s.) but reduced in all three tumor patients (2.23 +/- 0.55/1.23 +/- 0.43). The initial insulin response [microU/ml] in CP patients was also not altered after resection (19.7 +/- 17.3/16.0 +/- 18.2; n.s.) or after drainage (16.7 +/- 16.5/13.0 +/- 9.0; n.s.), whereas all three resected tumor patients showed reduced values (42.9 +/- 15.7/17.5 +/- 3.8). Stimulated C-peptide synthesis [ngmin/ml] was not substantially lowered in patients resected for CP (90.5 +/- 85.6/73.8 +/- 48.9; n.s.) or in the drainage group (121.3 +/- 67.5/98.0 +/- 57.2; n.s.), but this parameter was decreased in every tumor patient postoperatively (157.8 +/- 66.9/125.1 +/- 69.6). Resection in patients with chronic pancreatitis did not inevitably result in loss of beta cell function. Parenchyma-preserving drainage procedures had no measurable advantage in this respect.(ABSTRACT TRUNCATED AT 250 WORDS)

We reported previously the successful 72-hour cold rat pancreas preservation by using Perfluorochemical (PFC). The present study is to determine whether Fluorocarbon (FC) emulsion is as effective as PFC for long-term rat pancreas preservation. Lewis rat pancreases were stored in FC emulsion (4 degrees C) saturated by continuous supply of oxygen:carbon dioxide (95%:5%) (Group I) or by 100% pure nitrogen (Group II), or in PFC (4 degrees C) saturated by continuous supply of oxygen:carbon dioxide (95%:5%) (Group III) or nitrogen (Group IV) for 24 h and 48 h. Heterotopic pancreas transplantation into isogeneic diabetic rats were performed following preservation. Functional graft success rates following 24 h and 48 h cold storage were 71% (5/7) and 0% (0/5) in Group I, 71% (5/7) and 0% (0/5) in Group II, 100% (5/5) and 80% (4/5) in Group III, and 80% (4/5) and 0% (0/5) in Group IV, respectively. These results showed that, as an artificial blood substitute, the PFC with simple oxygen bubbling for 48-hour preservation of rat pancreas was much effective than FC emulsion, but not effective when saturated with nitrogen. We concluded that the PFC with saturated oxygen can obtain long-term successful preservation of rat pancreas. The direct oxygenation of the graft tissues is thought to play an important role in organ preservation.

Permselective acrylic membranes were employed to prevent immune rejection of discordant islet xenografts isolated from various large animals. Canine, porcine, and bovine islets were seeded into tubular diffusion chambers and transplanted into the peritoneum of 27 nonimmunosuppressed streptozotocin-induced diabetic Lewis rats. Six recipients received islet grafts from bovine calves, 7 received grafts from pigs, and 14 received grafts from dogs. Four of the latter were removed at 1 month. In the control group of 10 diabetic rats, 4 received nonencapsulated canine islets, 3 received nonencapsulated bovine islets, and 3 received nonencapsulated porcine islets. Recipients of encapsulated islets promptly dropped from a pretransplantation plasma glucose level of 487 +/- 36 (mean +/- SEM) to 84 +/- 2 (canine), 81 +/- 4 (bovine), and 81 +/- 3 mg/dl (porcine) during the first week. All of the animals sustained these levels for at least 1 month. One rat spontaneously reverted to diabetes at 54 days posttransplantation; 4 other rats became hyperglycemic (glucose, greater than 600 mg/dl) after membrane removal on day 30. The remaining 22 rats maintained fasting euglycemia for greater than 10 weeks. In contrast, rats that received nonencapsulated islets became hyperglycemic in less than 7 days. Intravenous glucose tolerance test K values (decline in glucose levels, %/min) at 1 month for the canine and bovine encapsulated islet transplant group were 3.5 +/- 0.3 and 3.3 +/- 0.1 compared with 3.3 +/- 0.1 (P = 0.63) and 0.91 +/- 0.1 (P less than 0.0001) for normal (n = 4) and diabetic (n = 4) control groups. Morphologic studies of long-term functioning grafts (30-130 days) revealed well-preserved alpha, beta, and delta cells, with varying degrees of granulation. These results demonstrate that immune isolation of islet tissue using permselective artificial membranes can protect discordant islet xenografts from immune rejection in the absence of any immunosuppressive drugs.

Improved glucose disposal after repeated intravenous (IV) glucose loads, known as the Staub effect, is absent in both type I and type II diabetes. Acute decrements of insulin secretion (AID) are observed following the early phase of insulin release on IV glucose stimulation in non-insulin-dependent diabetes mellitus (NIDDM). The AID cannot be corrected by acute infusions of glyburide or glipizide, or phentolamine, or by glycemic control after 2 weeks of intensive insulin treatments. Fifteen male subjects had 3 hourly successive IV glucose tolerance tests before and after 6 months of glipizide, at a final maintenance dose of 30 +/- 3 mg/d. Before treatment, AID, defined as the difference of the lowest of post IV glucose 7- or 10-minute samples from the preceding baseline insulin levels, was detected in the three loads: -6.4 +/- 1.9 microU/mL in the first, and then more consistently in the second and third loads, -11 +/- 2 microU/mL, and -17 +/- 9 microU/mL, respectively. There was a stepwise increase in insulin sums after each load. After glipizide therapy, the AIDS following all three IV glucose loads were no longer demonstrable; both insulin values, as well as insulin sums, were significantly elevated after all three glucose loads. While in the untreated state, the Staub effect was absent: serum glucose disappearances (K), corrected for glycosuria, were K1 = 0.52 +/- 0.02, K2 = 0.50 +/- 0.04, and K3 = 0.52 +/- 0.03.(ABSTRACT TRUNCATED AT 250 WORDS)

The endocrine function and histology of segmental pancreatic autografts with pancreatic exocrine diversion to the esophagus were assessed in nine dogs which survived longer than three years. An original twelve dogs underwent total pancreatectomy followed by a segmental pancreatic graft autotransplanted with pancreatic duct to esophagus anastomosis in the neck. All twelve dogs immediately had normal fasting glucose, nine of which sustained it for more than three years. One of the twelve dogs died on the tenth day from a thrombosis and two others died of causes unrelated to the graft; one of pneumonia and the other of an unknown cause, within the first year of transplantation. An intravenous glucose tolerance test performed three years after the transplantation revealed K values (1.90 +/- 0.37) which were not significantly different from those tested before the transplantation (1.92 +/- 0.42). The patency of the anastomosis between the pancreatic duct and the esophagus was clearly identified in the specimen of a dog sacrificed three years after the transplantation. The mucosa of the esophagus was macroscopically and microscopically almost normal. Histological studies of the autografts done three years after the transplantation showed almost normal pancreatic architecture in the islets and exocrine tissues, while histochemical analysis with immunoperoxidase stains confirmed the presence of insulin, glucagon and somatostatin. It is therefore possible that this new technique could be used for clinical segmental pancreatic transplantation.

Limits and possibilities of the transplantation of islets of Langerhans in pigs were studied. 6 x 10(4) to 3 x 10(6) islets and insulin producing fragments per pancreas were obtained by intraductal collagenase digestion of the pancreatic gland following total pancreatectomy. Islets grafted into the spleen or liver rendered normoglycemia to the pancreatectomized animals as demonstrated by normal fasting blood sugars and normal intravenous glucose tolerance tests as compared to not operated animals permitting a survival time of up to one year. Apancreatic controls died of ketoacidosis and diabetic coma 10 to 12 days posttransplant. The number of isolated and transplanted islets correlated well to the normoglycemic state of the animal. Beyond that the in vitro challenge of the islets with glucose and resulting insulin secretion was a very important indicator for the functional status and integrity of the islets after transplantation. Thus the pig appears to be a suitable model for the preclinical studying of islet transplantation especially since immunologic, physiologic and anatomic features of the pig are similar to those in the human regarding pancreas and nutrition.

Chronic pancreatitis was induced in 22 piglets by dividing all pancreatic attachments to the duodenum; five sham-operated piglets served as controls. Two piglets died of postoperative complications. The animals were autopsied 2, 4, or 6 weeks postoperatively. All operated animals developed chronic pancreatitis. Concomitant with the development of interstitial fibrosis, an increasing progressive atrophy of the exocrine parenchyma occurred, with preservation of the islets of Langerhans. This atrophy and fibrosis were considerable already after 2 weeks. In one piglet only there was some acute inflammation and fat necrosis, whereas all showed at least moderate chronic inflammation, which did not change with time. The growth of the piglets stopped, and all had diarrhoea, which was thought to reflect exocrine insufficiency. Two animals (9%) developed a large pancreatic pseudocyst, and all animals had wide pancreatic ducts. The endocrine function was undisturbed. Intravenous glucose tolerance tests showed that the animals did not become diabetic. This model is appropriate for the study of experimental pancreatitis.

Utilizing an intrasplenic canine islet autotransplant model, the effects of cold storage preservation on pancreatic tissue prior to and after collagenase dispersion were examined. A control series, in which freshly retrieved and prepared tissue was transplanted, yielded a 75% success rate (6/8). In contrast, when the pancreas was stored in modified silica gel filtered plasma (SGF I) for 24 hr, no autotransplant was successful (0/6). However, when the islet tissue was prepared following pancreatectomy and then stored in a mannitol-containing modification of SGF (SGF III), autotransplantation was successful in 83% (5/6) after 24 hr of preservation and in 60% (3/5) after 48 hr of preservation. Similarly, the islet tissue was stored in a hyperkalemic hydroxyethyl starch solution (HES) and this was successful in 20% (1/5) after 24 hr of preservation and in 50% (1/2) after 48 hr of preservation. Cold storage preservation techniques for the pancreas prior to islet isolation need to be refined, but dispersed islet-enriched pancreatic tissue can be successfully maintained at 4 degrees C for up to 48 hr prior to transplantation in dogs using established pancreas preservation solutions.

Preservation of the Lewis rat pancreas prior to islet isolation was accomplished by initial intraductal distension with the University of Wisconsin (UW) hydroxyethyl starch-lactobionate solution to which collagenase had been added, followed by simple cold storage at 4 degrees C for 0, 3, 12, 24, and 48 hr (n = 16-21 at each interval). The pancreases were then processed by digestion and mechanical dispersion to produce free islets of Langerhans. The mean islet yields (+/- standard errors of the means) were controls = 819 +/- 58 (n = 21), 3 hr = 867 +/- 51 (n = 20), 12 hr = 770 +/- 71 (n = 16), 24 hr = 805 +/- 62 (n = 18), and 48 hr = 722 +/- 55 (n = 16). None of these means differed significantly. The islets from pairs of donor pancreases (mean dose of islets = 1586 +/- 72) were transplanted intraportally into single isogeneic recipients with streptozotocin-induced diabetes (plasma glucose greater than 400). The preservation interval directly influenced the outcome of these islet isografts in the following manner: (i) Rates of functional success (nonfasting glucose less than 200 mg/dl) were 100% after storage times of 0 hr (n = 10), 3 hr (n = 8), and 12 hr (n = 8); 86% with a storage time of 24 hr (n = 7); and 0% after 48 hr (n = 8). (ii) Return of euglycemia was increasingly delayed with increasing preservation intervals.(ABSTRACT TRUNCATED AT 250 WORDS)

Despite the frequent use of in vitro tissue culture before islet cryopreservation, no study has evaluated the ability of this procedure to improve the recovery or in vivo function of frozen-thawed islets. To evaluate this, quantities of 2500 Wistar-Furth (WF) rat islets were allocated to each of four groups (n = 8 each): group 1, freshly isolated; group 2, 48 hr in vitro culture; group 3, cryopreservation; group 4, cryopreservation after 48 hr in culture. Islets were frozen slowly at 0.25 degrees C/min and thawed rapidly at 200 degrees C/min. The number of islets recovered after culture or cryopreservation was determined and viability was assessed by measuring weekly indices of plasma glucose (PG), urine glucose (UG), urine volume (UV), and weight after implantation into the portal vein of streptozotocin-diabetic WF recipients. Islet recovery was 97% after culture, 95% after cryopreservation, and 94% after culture-then cryopreservation. After implantation of group 1 and 2 islets, PG was less than 150 mg/dl at 1 week and UG and UV were normal by 1-2 weeks. Group 3 islets restored normoglycemia at 3 weeks and other indices of diabetes were reversed by 4 weeks; group 4 islets restored normoglycemia at 4 weeks and indices returned to basal after 4 weeks. At intravenous glucose tolerance testing (ivGTT), the K values (mean decline in glucose, %/min, +/- SE) were group 1, 1.6 +/- 0.3; group 2, 1.5 +/- 0.3; group 3, 0.6 +/- 0.1; and group 4, 0.7 +/- 0.2. These data show that cryopreservation preserves freshly isolated or cultured islets that reverse the indices of diabetes after implantation.(ABSTRACT TRUNCATED AT 250 WORDS)

Clinical pancreatic islet transplantation has been impeded by the inability to isolate an adequate mass of functional tissue that will ameliorate diabetes. A simplified method of canine islet isolation was developed that allowed for either intrasplenic or intrahepatic transplantation. Following total pancreatectomy, parenchymal digestion was accomplished by intraductal collagenase perfusion and stationary incubation. The digested tissue was dispersed by filtration through a steel mesh (400 microns), washed, and separated on a discontinuous dextran density gradient. Enhanced islet tissue (2-4 ml) was recovered from the uppermost interface of the gradient and autotransplanted. The islet isolation procedure was tested in two series of dogs undergoing either intrasplenic or intrahepatic engraftment. Immediate and sustained normoglycemia (plasma glucose less than 200 mg%) was obtained in 5 of 8 dogs (63%) in the intrasplenic group and 6 of 8 dogs (75%) in the intrahepatic group. The mean fasting plasma glucose concentration 2 weeks after transplantation was 102.8 +/- 6.4 mg% in the intrasplenic group and 103.3 +/- 8.4 mg% in the intraportal group. The mean IVGTT K-values 2 weeks after transplantation were -1.41 +/- 0.35% and -1.21 +/- 0.13%, respectively. On the basis of insulin content, the islet yield was 33.0 +/- 3.7% of the total pancreas in the intrasplenic group and 33.0 +/- 3.1% in the intrahepatic group. Islet mass was enhanced 10.2 +/- 1.5 and 20.0 +/- 6.2 fold, respectively, on the basis of insulin/amylase ratios. The success rate in this canine model compared favorably with previously published results from other laboratories.(ABSTRACT TRUNCATED AT 250 WORDS)

20 dogs underwent total pancreatectomy. Pancreatic fragments were produced using an intraductal collagenase perfusion technique and mechanical disruption of the pancreatic gland. The resulting tissue suspension was transplanted as an autotransplant to the spleen of each animal by intravenous reflux injection into the splenic vein. In 75% of all transplanted animals long-term function with normoglycemia was restored while i.v. glucose tolerance tests were impaired as compared to 13 controls. The spleen insulin content of these successfully transplanted animals corresponded to only 5 to 15% of the original pancreas insulin content which was responsible for the reduced function. Intraductal collagenase perfusion is a reliable method to restore normoglycemia after total pancreatectomy in the autotransplant model using pancreatic fragments. To improve the metabolic function of the islet transplant an increased number of islets has to be implanted.

One problem preventing islet transplantation becoming a clinical reality in the treatment of insulin-dependent diabetes is the unsatisfactory, low yield processes for harvesting islets. This study was designed to find the most effective harvesting process and to examine the feasibility of obtaining more than one graft from a single pancreas. After confirming that normoglycaemia at one month could be achieved in dogs using 50 per cent of an autograft prepared by a previously established method, novel techniques were assessed using 33 per cent of the tissue obtained. An isolation process involving perfusion of the duct by enzymes allowed normoglycaemia using only one-third of the graft preparation and these animals exhibited significantly improved glucose handling and insulin output when compared with recipients of grafts prepared by the established method. A significant improvement in harvesting technique has, therefore, been demonstrated and this will exploit the potential of multiple donor grafts in islet transplantation and the method may be worthy of trial in the human gland.

Storage of pancreatic islets by cryopreservation would greatly facilitate a large scale program of clinical islet transplantation. We report success on long-term follow-up with autotransplantation of frozen/thawed canine pancreatic fragments. Total pancreatectomy and islet isolation by collagenase ductal perfusion and mechanical disruption preceded either acute autotransplantation or cryogenic preservation prior to autotransplantation. Cryopreservation was by dimethylsulfoxide equilibration, cooling at 0.25 degrees C/min to -75 degrees C, storage in liquid N2 and thawing at 3.5 degrees C/min. Four of five acutely autotransplanted dogs remained normoglycemic for 20 months, with three of four maintaining normal K values on intravenous glucose tolerance test (IVGTT) and nondiabetic values on oral GTT. Four of four dogs transplanted with frozen/thawed islets remained normoglycemic for 15 months with three of four maintaining nondiabetic IV GTT K values and normal oral GTTs for 15 months. Both acutely transplanted and frozen/thawed islets are capable of maintaining long-term metabolic control. Cryopreservation preserved viability of sufficient canine pancreatic islets to reverse diabetes with autotransplantation. Function of the frozen-thawed islets showed minimal deterioration during a follow-up of 15 to 18 months.

Acceleration of glucose tolerance after repetitive intravenous glucose loads (Staub-Traugott effect) has not previously been tested in non-insulin-dependent diabetes (NIDDM). Six overt, untreated subjects were administered three hourly IV glucose tolerance tests (GTT). The glucose disappearance rate (K) changed very little between loads, indicating a suppressed Staub effect. However, insulin levels increased with each load. The characteristic loss of early phase insulin release after the first intravenous glucose injection was recovered with the third injection. After four months of tolazamide treatment the fasting plasma glucose fell from 180 +/- 19 to 134 +/- 13 mg/dL. Despite dramatic potentiation of glucose-stimulated insulin release and further improvement of early phase insulin release, K values again failed to progressively rise. This paradox occurred even in three additional subjects tested after two years of tolazamide treatment, suggesting that tolazamide may not ameliorate the postreceptor defects that impede the expression of the Staub effect. The applicability of the glucose facilitatory effect to the treatment of NIDDM might be limited to subjects in whom adjunctive measures have reestablished effective tissue responsiveness to endogenous insulin.

Allograft rejection is the major obstacle to clinical pancreatic islet transplantation. We attempted Cyclosporine A (CsA) immunosuppression of intrasplenic pancreatic dispersed fragment allografts in unrelated, unmatched dogs. Animals underwent (1) pancreatectomy only, (2) autotransplantation, or allotransplantation with (3) no immunosuppression (NIL), (4) azathioprine (AZA 3 mg/kg/day) and prednisone (PRED 2 mg/kg/day), or (5) CsA (25 mg/kg/day). Graft preparation was by collagenase ductal perfusion and mechanical disruption and transplantation was by splenic venous reflux. Rejection was defined by permanent hyperglycemia (greater than 150 mg/dl). Apancreatic dogs survived 6.0 +/- 0.5 days; autotransplanted dogs remained normoglycemic at 6 months. Rejection occurred at NIL, 5.0 +/- 0.6 days; AZA/PRED, 1.8 +/- 0.3 days; and CsA, 19.3 +/- 5.6 days. Survival was NIL, 16.0 +/- 3.4 days; AZA/PRED, 13.4 +/- 1.4 days; and CsA, 33.3 +/- 4.6 days. CsA showed significant advantage over both groups in both time to rejection (P less than 0.05) and survival (P less than 0.01). CsA toxicity was minimal. CsA achieved significant delay in rejection and improved survival in unmatched, unrelated dogs after intrasplenic allotransplantation with pancreatic dispersed fragments.

Islet cell transplantation is a potential treatment for insulin-dependent diabetes (IDD), assuming that physiological hormone release results in biochemical normality which, in turn, reduces the incidence of long-term diabetic complications. These experiments sought to create a metabolic model of IDD in a large animal and investigate the effects of insulin therapy and islet transplantation. After demonstrating the success of transplantation, further studies examined the feasibility of creating multiple grafts from a single donor and their long-term preservation. The totally pancreatectomized dog, treated with suboptimal insulin therapy was biochemically identical to human IDD and, when more appropriate doses of insulin were administered, metabolic improvements occurred without restoring normality. Islet cell autotransplants rendered such animals metabolically normal apart from persisting impairment of glucose tolerance. The isolation technique permitted the production of at least two recipient grafts and some success was achieved after graft cryopreservation. The experiments confirm the theoretical background to islet transplantation and demonstrate its applicability in a large animal model before allotransplantation.

(1) The freezing protocol we have devised for rat islets results in normal clinical indices and almost normal glucose tolerance in diabetic recipients of the same inbred strain. (2) Cryopreservation of canine islet-containing pancreatic tissue required a higher temperature than rat islets during exposure to the protective agent. (3) Because of the similar compactness of the pancreas in man and dog, we consider this canine model useful for formulating optimal cryopreservation techniques for the human pancreas. (4) Cryopreservation may partially purify islet-containing tissue of its exocrine content.

Neonatal rat pancreatic tissue was frozen to -196 degrees C using Me2SO as a cryoprotectant and a slow freezing rate to -70 degrees C followed by immersion in liquid nitrogen. Rapid thawing was used. Viability was demonstrated by successful transplantation to streptozotocin-induced diabetic recipients. Long-term preservation, up to 4 weeks, did not demonstrably affect viability. Cryopreservation techniques may afford a method for providing a diabetic recipient the opportunity to receive a large quantity of pooled islet tissue from well-matched donors.

1. Glucagon tolbutamide, either alone or in combination, were injected i.v. into pigs and the effect upon plasma glucose and insulin concentrations measured. 2. Glucagon gave similar insulin responses to those seen in humans, but insulin responses to tolbutamide were less than in humans. 3. Combined doses of glucagon and tolbutamide gave similar, though reduced, responses to those seen in humans. At the highest combined doses applied, glucose concentration remained reduced for up to 6 hr. The insulin responses were approximately equal to the sum of the responses to each substance given alone.

Forty-nine dogs were made diabetic by total pancreatectomy. Fifteen untreated pancreatectomized animals survived a mean (+/-S.E.) of 7.0 +/- 1.1 days with a mean (+/-S.E.) plasma glucose level of 402 +/- 26 mg/100 ml before death. The pancreata of 32 dogs were distended with cold (4 degrees ) Hanks' solution, minced, digested with collagenase (600 U/ml tissue) for 15-25 minutes, and autotransplanted either into the splenic artery (three dogs), directly into the splenic pulp (21 dogs), or into the portal vein (ten dogs). Tissue infusion into the splenic artery resulted in infarction and persistent hyperglycemia. Direct implantation into the splenic pulp of tissue digested for 15, 20 and 25 minutes resulted in permanent normoglycemia (fasting plasma glucose < 150 mg/100 ml) in 7 of 8, 7 of 7, and 6 of 6 dogs respectively. Glucose tolerance test mean (+/-S.E.) K values (% decline of plasma glucose concentration/minute) in these groups two weeks after transplantation were 1.20 +/- 0.20%, 1.60 +/- 0.25 and 0.70 0.08% respectively, indicating that 20 minutes digestion was best for intrasplenic transplantation. Tissue prepared in the optimal manner (20 minutes digestion) and embolized into the liver resulted in normoglycemia in three of eight dogs, and a mean (+/-S.E.) K value of 0.77 +/- 0.10%. Both dogs receiving tissue dispersed for 25 minutes into the portal vein remained hyperglycemic. In the dogs subjected to intraportal transplantation, portal pressure rose from a mean (+/-S.E.) of 6.5 +/- 0.6 cm H(2)O before to 21.9 +/- 2.2 cm H(2)O immediately after tissue embolization, but declined to 6.5 +/- 1.0 cm H(2)O by ten weeks in animals becoming normoglycemic. We conclude that in dogs direct implantation of pancreatic tissue into the splenic pulp is superior to embolization into the portal vein or splenic artery because the splenic circulation is not compromized, portal hypertension is obviated, and glucose metabolism is best controlled as judged by glucose tolerance test K values.

A single subdiabetogenic dose of alloxan administered to the weanling rat induces a persistent state of latent diabetes which progresses to fasting hyperglycemia by the seventh generation. Initial descendants of alloxan-treated animals have hyperinsulinism which progresses to insulinopenia in later generations. Later generation animals develop ketoacidosis when challenged with a dose of alloxan that has no effect on control animals. The significant sex difference in glucose tolerance rates disappears as the animals become more diabetic and decreased fertility and parity become apparent. One explanation for this data remains the hypothesis of paramutation, induced by alloxan, affecting regulator gene activity. Light microscopy of diabetic animals shows no pathology.