Search

It is still a controversial question whether insulin suppresses its own secretion. We prepared pure human islets from three pancreases by collagenase digestion and density gradient purification. Aliquots of 200 islet equivalents (IE, 150-μm sized-islets) were sequentially perifused at 37°C with 3.3 mmol/l glucose (3.3G, 40 min), 16.7 mmol/l glucose (16.7G, 30 min) and again 3.3G (30 min) after 24 h, 37°C culture in CMRL 1066 medium with or without the addition of either 200 or 400 μU/ml human insulin in the incubation medium (6 replicates each). Insulin secretion was assessed by C-peptide (Cp) measurement in the perufusate. Without added insulin (C) and with 200 (Ins200) or 400 (Ins400) μU/ml added insulin, basal Cp release was 0.12±0.03, 0.14±0.02 and 0.14±0.04 ng/ml, respectively. At 16.7G, the first-phase secretion peak (expressed as Cp value) was significantly lower with Ins200 (0.47±0.13 ng/ml,P

INCE 1985,27 transplants have occurred at Washington University School of Medicine (WUMS) in St. Louis and 4 transplants in conjunction with the University of Western Ontario, London, Ontario. The majority of suc- cessful transplants have occurred since 1989 when we entered into the era of better immunosuppression, early intensive insulin therapy, purer preparations, immunoalter- ation by 24°C culture, and cryopreservation resulting in larger transplanted islet preparations. The two groups of patients most extensively followed at WUMS were patients with established kidney grafts followed by islet transplant (EKI, n = 8) and those receiving simultaneously trans- planted kidney and islet transplant (SKI, n = 9). Several controversies concerning islet transplantation still exist. For example, should islet transplantation occur simultaneous with or following renal transplant? Does cryopreservation and/or islet culture make a difference in islet function? What is the role of human leukocyte antigen (HLA) matching? Does islet mass improve or hamper long-term islet function? Presented here are the results of a retrospec- tive study addressing these questions in regard to our EKI and SKI patients.

We have created a new transgenic model in which the human Epstein-Barr virus receptor, CR2 (CD21), has been expressed in pancreatic beta-cells. Mice derived from three transgenic founders bred into H-2b (C57BL/6) or H-2k (CBA) genetic backgrounds did not become spontaneously diabetic. After transplantation of CR2-expressing islets under the kidney capsule of genetically matched recipients, a histologic picture of peri-insulitis was found. However, animals did not manifest cellular invasion of the islets, destruction of the islets, or diabetes for at least 230 days. Significant numbers of both T and B lymphocytes were found in the cell population surrounding the islets. A pronounced serologic response to CR2 was also present and appeared to precede the onset of peri-insulitis. Thus, in this model, we have separated the process of diabetes induction into at least two phases. One is associated with peri-islet infiltration and an antibody response. However, at least one second signal is likely necessary for the process of islet destruction to follow.

Up-regulation of maternal islet function is essential to accommodate the increased demand for insulin during pregnancy. Previously, we suggested that lactogenic activity regulates islet function during pregnancy. However, this hypothesis was based on the effect of homologous PRLs on islets, since the homologous placental lactogens (or islets) were unavailable. In this study we examine the direct effects of homologous placental lactogens (PL), PRL, and GH on insulin secretion and B-cell division in rat, mouse, and human islets in vitro. Neonatal rat islets were cultured for 8 days in the presence of 0-1000 ng/ml rat PL-I (rPL-I), rPRL, or rGH. Media were changed daily, and the insulin concentration was determined. rPL-I and rPRL (500 ng/ml) treatment resulted in a 2-fold increase in insulin secretion. rGH (1000 ng/ml) elicited a 30% increase in insulin secretion. Similarly, cell replication, as indicated by BrdU incorporation into B-cells, was increased 4-fold in the presence of rPL-I and rPRL. The ED50 for insulin secretion and 5'-bromo-2'-deoxyuridine (BrdU) incorporation was 70 ng/ml for rPL-I and 150 ng/ml for rPRL. Similarly, in adult rat islets, insulin secretion was increased 1.6-fold, and B-cell replication increased 3-fold in the presence of the lactogenic hormones. Neonatal mouse islets were cultured for 8 days in the presence of 500 ng/ml mouse (m) PL-I, mPL-II, mPRL, or mGH. mPL-I, mPL-II, and mPRL treatment resulted in a 2-fold increase in insulin secretion. mGH elicited a 30% increase in insulin secretion. BrdU incorporation into B-cells was increased 3-fold in the presence of mPL-I and mPRL and 2-fold in the presence of mPL-II. Adult human islets were cultured for 8 days in the presence of 1 microgram/ml human (h) PL, hPRL, or hGH. For human islets isolated from six pancreata obtained from females, hPL (138 +/- 10%), hPRL (133 +/- 9%), and hGH (117 +/- 3%) significantly increased insulin secretion compared to that from control islets. This study compares the direct effects among homologous PLs, PRLs, and GHs on insulin secretion and B-cell division in rat, mouse, and human islets. The results indicate that placental lactogen directly regulates islet function in several species and is probably the principal hormone responsible for the increased islet function observed during normal pregnancy.

In vitro culture of rat islets at 24 degrees C for 7 days in tissue culture medium CMRL 1066 almost completely eliminated lymphoid cells from the islets. Immunostaining of the islets with monoclonal antibody OX4 for demonstration of class II major histocompatibility complex (MHC)-expressing cells revealed a decrease from 13.1 +/- 0.6 positive cells per islet on day 0 to 0.7 +/- 0.1 cells per islet on day 7. A comparable decrease was found using OX1 for demonstration of all leukocytes. In contrast, culture of rat islets at 24 degrees C for 7 days with tissue culture Roswell Park Memorial Institute (RPMI) 1640 medium was not as effective in eliminating lymphoid cells as in medium CMRL 1066 (3.0 +/- 0.2 class II MHC positive cells per islet at 7 days). Effective elimination of intraislet lymphoid cells apparently is due to the combined effect of low temperature culture and the tissue culture medium CMRL-1066. The second goal of the study was to determine whether the destructive effect of interferon gamma (IFN-gamma) on rat islets in culture was due to intraislet lymphoid cells. In vitro culture of rat islets with IFN-gamma (1000 units/ml) at 37 degrees C caused almost complete destruction of the islets at 7 days. If intraislet lymphoid cells were eliminated from the islets by in vitro culture at 24 degrees C followed by exposure to IFN-gamma (1000 units/ml) for 7 days at 37 degrees C, then IFN-gamma did not cause destruction of the islets and transplants of the treated islets produced normoglycemia in diabetic recipient mice. These findings indicate that intraislet lymphoid cells are responsible for destruction of islet cells when these cells (presumably macrophages) are activated by IFN-gamma. Intraislet lymphoid cells may play a significant role in destroying islet cells in autoimmune diabetes.

Previous studies have suggested that pancreatic islet glucose transport is mediated by a high-Km, low-affinity facilitated transporter similar to that expressed in liver. To determine the relationship between islet and liver glucose transporters, liver-type glucose-transporter cDNA clones were isolated from a human liver cDNA library. The liver-type glucose-transporter cDNA clone hybridized to mRNA transcripts of the same size in human liver and pancreatic islet RNA. A cDNA library was prepared from purified human pancreatic islet tissue and screened with human liver-type glucose-transporter cDNA. We isolated two overlapping cDNA clones encompassing 2600 base pairs, which encode a pancreatic islet protein identical in sequence to that of the putative liver-type glucose-transporter protein. Xenopus oocytes injected with synthetic mRNA transcribed from a full-length cDNA construct exhibited increased uptake of 2-deoxyglucose, confirming the functional identity of the clone. These cDNA clones can now be used to study regulation of expression of the gene and to assess the role of inherited defects in this gene as a candidate for inherited susceptibility to non-insulin-dependent diabetes mellitus.

Monolayer cultures of adult rat islets were used for cinemicrographic studies on the movement of beta granules in the cytoplasm of beta cells. Stimulation of insulin release by glucose produced a significant increase in the amount of beta granule movement in the cultured cells. Disruption of the microtubular system with vinblastine or stabilization of microtubules with deuterium oxide inhibited the induction of granule movement by glucose. Incubation of the cultures in the absence of calcium inhibited glucose-induced movement of beta granules, whereas the subsequent addition of calcium permitted movement to occur. The addition of epinephrine also inhibited glucose-induced beta granule movement. In the presence of nonstimulating levels of glucose, movement of beta granules was present. The amount of movement varied from cell to cell suggesting a difference in base line activity of individual beta cells. The movement of individual beta granules was saltatory in type, and the average rate of movement was 1.5 mum. per second. The study demonstrated that directed movement of beta granules occurs following stimulation with glucose and adds further support to the hypothesis that the microtubular-microfilamentous system is involved in the intracellular translocation of beta granules to the cell surface where they are released by emiocytosis.

Three weekly intraperitoneal injections of complete Freund's adjuvant (CFA) and, one day later, 25 mg/kg of STZ have been reported to cause a gradual onset of autoimmune diabetes. We have previously reported that the onset of diabetes in this model occurs too rapidly to be due to a specific immune reaction and that other compounds that activate macrophages also potentiate low-dose STZ in rats. We have proposed that mediators released from activated macrophages may enhance the cytotoxic effect of STZ. In the present study, we have observed that intraperitoneal injections of silica, a macrophage toxin, markedly decrease the incidence and severity of diabetes induced with CFA and low-dose STZ.

The findings in this series of investigations indicate that the passenger leukocyte concept applies to islet transplants. Six methods have been developed which prevent rejection of islet allografts in rats and mice without requiring the continuous use of immunosuppressive drugs in the recipient. Initial studies indicate that the passenger leukocyte concept also applies to the prevention of rejection of islet allografts in the dog. Thus the problems remaining with respect to human application are two. One is to determine which of the six methods will completely prevent rejection of islet allografts in dogs and could serve as a model for human application. The second is to modify and adapt the Velcro technique and the automated procedure for mass isolation of islets to the human pancreas. When we have been successful in modifying these isolation procedures so that we can obtain at least 100-150,000 islets, then human islets will be transplanted into the omentum of diabetic subjects who have received a kidney transplant and are already being treated with immunosuppressive agents. The purpose of these initial studies in humans will be to determine whether a sufficient mass of endocrine islet tissue has been transplanted to maintain normoglycemia and normal carbohydrate metabolism in these individuals. We hope that by the time these studies are completed we will have finished the studies in dogs on the selection of the optimal method for preventing rejection and will then be able to initiate human islet transplants without the use of immunosuppressive drugs. Dr. Davie and I also believe that these methods may be applicable to the prevention of rejection of other tissues and organs. It should be quite easy to apply these procedures to transplants of the parathyroid. Dr. Lafferty already has evidence that parathyroid tissue is present in established allografts of thyroid in mice following in vitro culture of the thyroid in 95% O2. It is possible that the procedures will also apply to other endocrine tissue such as the adrenal, the ovary and neuroendocrine tissue. In addition, some of the procedures that have been developed recently could be used for perfusion of organs such as the heart, kidney and liver before transplantation to determine whether alteration or removal of passenger leukocytes in these large organs would affect the survival of the allografts. It is apparent that these past few years have been extremely exciting and we only hope that the next few years will be even more exciting.

Alloxan was found to inhibit a Ca2+- and calmodulin-dependent protein kinase recently identified in pancreatic islets. This effect of alloxan may be specifically related to the inhibitory action of alloxan on insulin secretion from islets since: 1) in islet-cell subcellular fractions, alloxan at micromolar concentrations irreversibly inhibits the Ca2+- and calmodulin-dependent protein kinase activity; 2) pretreatment of intact islets with alloxan at concentrations that inhibit insulin secretion similarly inhibits the protein kinase activity; and 3) alloxan inhibition of both insulin secretion and protein kinase activity in intact islets can be prevented by D-glucose. This inhibition by alloxan appears to be a direct effect on the enzyme since alloxan treatment of either the islet homogenate or the microsomal fraction enriched in protein kinase activity inhibited the kinase activity with similar concentration dependence. These results suggest that alloxan-induced inhibition of a Ca2+- and calmodulin-dependent protein kinase may represent a critical inhibitory site which mediates alloxan-induced inhibition of insulin secretion.

This report describes a method for monolayer cell culture of adult rat pancreatic islets. In this procedure, the isolated islets of Langerhans were enzymatically dispersed and subsequently cultured for 2 to 3 weeks. Enhanced secretion of insulin was present over the basal secretion rate when the cultures were exposed to a high glucose concentration alone or to a combination of glucose, theophylline and glucagon. The ultrastructural studies of the beta cells in culture demonstrated preservation of their morphological characteristics throughout the culture periods.

Active calcium uptake was demonstrated in a subcellular fraction of islets which was enriched in endoplasmic reticulum. Calcium uptake was stimulated by ATP in a magnesium-dependent manner. The rate of calcium accumulation was sustained by oxalate (10 mM) and uptake was prevented or reversed by addition of the calcium ionophore A23187. This calcium uptake process was not affected by azide or ruthenium red. Direct comparison of calcium uptake by endoplasmic reticulum-enriched and plasma membrane-enriched fractions indicated that the uptake was not due to contamination of the fraction with plasma membrane vesicles. These factors as well as the purity of the fraction indicate that the calcium uptake system resides in the endoplasmic reticulum. The properties of the islet endoplasmic reticulum calcium uptake system are similar to properties reported for endoplasmic reticulum derived from other cell types. These properties include stimulation by potassium and a Km for ionized calcium of 1.5 +/- 0.3 microM. The islet-cell endoplasmic reticulum may play a critical role in cellular calcium homeostasis and contribute to the regulation of insulin secretion.

The effect of cyproheptadine on the structure and function of rat pancreatic beta cells was studied in vitro by culturing isolated islets in media containing the drug (0.5 mM). After 8 days in culture the ultrastructure of islet cells maintained in control media appeared well preserved, being similar to the previously reported for islets kept in long term organ culture. In contrast beta cells from islets incubated in media containing cyproheptadine appeared degranulated and the rough endoplasmic reticulum showed cisternal dilation and vacuole formation. These vacuoles were filled with an electron-dense granular material and their surface was usually studded with ribosomes. These lesions are identical with those produced by the administration of cyproheptadine to rats in vivo. In addition to these findings, increased numbers of lysosomes and myeloid bodies were observed in both alpha and beta cells. Compared with that of the controls, the 24-hour basal insulin secretion of islets cultured in the presence of cyproheptadine was significantly reduced from the 4th day of the study onwards. It is thus concluded that cyproheptadine has direct effects on the morphology and function of the rat pancreatic beta cell.

Aberrant Ia antigen expression has been implicated in the pathogenesis of type 1 diabetes. Ia antigen expression was induced on isolated B10.BR murine islet parenchymal cells by culturing them for 5 days with lymphokine supernatants containing interferon-gamma (IFN-gamma) or with recombinant murine IFN-gamma + recombinant tumor necrosis factor. Ia positivity was confirmed by indirect immunofluorescence. Islets cultured for 5 days without cytokines were Ia-negative. Purified B10.BR islets allografted into the portal veins of C57BL/6J mice do not reject, which allowed the authors to determine whether aberrant expression of Ia on parenchymal cells has a deleterious effect on allograft survival. Ia-positive or Ia-negative islets were transplanted via the portal vein into diabetic C57BL/6J mice. All mice remained normoglycemic until they were killed at 30 to 60 days. Well-granulated islet allografts were identified histologically in all mice. The experiment was repeated using Balb/cJ mice as donors. Purified Balb/cJ islets are rapidly rejected by C57BL/6J mice. Induction of Ia expression on Balb/cJ islets significantly improved allograft survival. These findings indicate that Ia-positive islet cells do not induce rejection in these allograft models but may actually have a protective effect.

The purpose of this study was to determine the effect of pancreatic islets on the survival of clusters of rat hepatocytes transplanted across a major histocompatibility barrier into an ectopic site. Aggregates of hepatocytes and pancreatic islets were obtained from Wistar-Furth rats by an automated method of collagenase digestion. Approximately 8000 Wistar-Furth hepatocyte clusters (100 to 300 micron diameter) were transplanted beneath the renal capsule of Lewis rats with or without the addition of 800 pancreatic islets. Recipients received subcutaneous injections of cyclosporine A (CsA): 30 mg/kg on days 0, 1, and 2, 10 mg/kg on days 3 to 7, and 10 mg/kg every other day for days 8 to 28. In the absence of CsA recipient treatment, all the allografts were rejected within 7 days. The combined hepatocyte-islet allografts in recipients treated with CsA showed maintenance of morphologic integrity of both hepatocytes and islet cells 1, 2, and 4 weeks after transplantation, whereas in the absence of pancreatic islets, the hepatocellular aggregates degenerated despite CsA treatment, leaving only a thin rim of hepatocytes immediately adjacent to the renal parenchyma. It can be concluded that in immunosuppressed recipients hepatocyte-islet renal subcapsular allografts survived despite the presence of an intact liver, which indicates a possible role of trophic factors released locally from the islets.

Islets were isolated from human pancreases by the distention method and purified by centrifugation on Ficoll or Percoll gradients. The purity of the final preparations was 60% to 90% islets, making it possible to culture the islet preparations in vitro. Perifusion of the islet preparations after 1, 3, or 7 days of culture at 24 degrees C indicated that the islets were viable and would respond to glucose stimulation. If the islets were returned to 37 degrees C culture for 1 day, then the insulin secretory response at 7 days was comparable with the response during the first day of culture at 37 degrees C. Extending the culture period at 24 degrees C to 14 days damaged the islets as indicated by the lack of response to glucose stimulation. The Ficoll technique was a much more effective method for purifying the islets, since it provided approximately twice the yield of islets and insulin compared with the Percoll procedure. The mean yield of purified islets by the Ficoll technique was 2180 +/- 325 islets/gm of pancreas with a calculated islet mass of 3.7 +/- 0.8 mm3/gm. The findings that massive numbers of purified human islets can be obtained by centrifugation on Ficoll gradients and that the purified islet preparations remain morphologically and functionally intact during 7 days' culture at 24 degrees C make it possible to assess the function of donor islets before human islet transplantation, transplant the islets via the portal vein, and use low-temperature culture as a possible approach for altering the immunogenicity of donor human islets.

Individual rat islets could be aggregated into single megaislets in vitro and the megaislets remained morphologically and functionally intact after a 7-day period of culture in the presence of 95% O2 and 5% CO2. Cultured rat megaislets transplanted beneath the renal capsule of diabetic mice produced normoglycemia in the recipients and the survival of the xenografts was markedly prolonged by the 7-day exposure of a high oxygen tension. A single injection of antilymphocyte sera to mouse and rat lymphocytes into the recipients receiving cultured megaislets did not produce a further increase in the percentage of survival of the grafts at 70 days after transplantation. Lymphoid aggregates were present around xenografts of cultured negaislets at 60 and 90 days after transplantation. This lymphoid reaction did not interfere with the function of the xenografts since the recipients were normoglycemic and removal of the grafts resulted in a rapid return to a diabetic state. Intraportal and intrasplenic transplants of cultured rat megaislets did not survive as long as the xenografts of megaislets transplanted beneath the renal capsule. The renal subcapsule site apparently provided some immunological advantage for delaying acute rejection since transplants of individual, fresh rat islets survived for twice as long under the renal capsule as compared wtih intraportal transplants of fresh rat islets.

The uptake of theophylline and caffeine was determined in isolated pancreatic islets employing a dual isotope procedure with sucrose as an extracellular marker. Islets rapidly accumulated caffeine and theophylline with apparent dissociation constants of approximately 23 and 6 mM, respectively. Theophylline inhibited the uptake of caffeine and caused displacement of caffein from islets. These results indicated a competition by theophylline and caffeine for a common site (binding and/or transport carrier). In addition, theophylline and caffeine inhibited D-glucose transport in a dose-dependent manner and within the limits of the experimental system, this inhibition appeared to be non-competitive. (Bu)2cAMP under similar experimental conditions exerted no effect on D-glucose transport. These results present evidence for a rapid uptake of theophylline and caffeine in pancreatic islets, which is compatible with their immediate cellular effects. In addition, these results demonstrate a direct effect by theophylline and caffeine on D-glucose transport which appears independent of their ability to alter intracellular cAMP levels.

Islets from male B10.BR mice (H-2k) were isolated by the collagenase technique, hand-picked with a Pasteur pipette, and incubated in tissue culture media supplemented with recombinant murine interferon-gamma (rIFN-gamma), recombinant murine tumor necrosis factor (rTNF), or both. IAk-molecules could be identified on the surface of islets incubated for 5 days with a combination of rIFN-gamma (1, 10, or 100 ng/ml) and rTNF (10 or 50 U/ml) by indirect immunofluorescence. Optimal concentrations of rIFN-gamma and rTNF, 10 ng/ml and 50 U/ml, respectively, were used in all subsequent experiments. Weak Ia-positivity could be identified on the surface of islets cultured with both cytokines for as little as 48 hours; however, the staining appeared most intense after 5 days of culture. Intensely Ia-positive islets were then carefully washed and cultured in media without either cytokine; Ia positivity could be identified on the surface of these islets for up to 1 week. Dispersed islet cells were cultured with rIFN-gamma alone (10 ng/ml), rTNF alone (50 U/ml), or both cytokines for 5 or 10 days. After either 5 or 10 days of culture with both cytokines, intense immunofluorescent staining for Ia could be identified on the surface of greater than 80-90% of the viable islet cells. Culture with IFN alone for 10 days resulted in 15-20% Ia positivity; culture with TNF alone did not cause Ia expression.

The distribution of radioautographic grains over organelles within the beta cells of rat islets of Langerhans was investigated at various times after pulse labeling of the isolated islets with tritium-labeled amino acids. Ten minutes after the start of labeling most of the grains were situated over the endoplasmic reticulum and cytoplasm; by contrast, 60 min from the start of labeling the majority of the grains were associated with the beta granules. At 20, 30, and 45 minutes after pulse labeling the proportion of grains associated with the Golgi complex was increased two- to three-fold over the 10- or 60-minute values. The distribution of radioautographic grains over granules in the intact cells did not suggest that the electron-lucent type of secretory granules were precursors of the electron-opaque granules. Furthermore, studies of the pattern of grains over granules isolated by centrifugation 60 min after pulse labeling showed no preferential labeling of the electron-lucent type of granule. It is concluded that labeled amino acids are incorporated initially in the endoplasmic reticulum, and that the label subsequently appears in the beta granules. The Golgi complex participates either in the formation of the beta granule or in the translocation of the granule through the cytoplasm of the cell.