Ganglioside expression in human pancreatic islets

Recent biochemical studies have shown that the cytoplasmic islet cell-antibody autoantigen has properties of a monosialoganglioside (GM). To characterize islet glycolipids and ascertain whether islets express unique gangliosides, we determined the pattern of ganglioside expression in whole human pancreas and isolated human islets using high-performance liquid chromatography (HPLC) and high-performance thin-layer chromatography (HPTLC). The major gangliosides detected in glycolipid extracts of whole human pancreas were GM3, GD3 (disialoganglioside), and in a lesser amount, a GD1a-comigrating ganglioside. In contrast to whole human pancreas, isolated human islets were found to predominantly express GM3, an acidic glycolipid comigrating with GM2, and a ganglioside with mobility between GM2 and GM1 by both HPLC and HPTLC. Quantitation of the major ganglioside UV peaks seen on HPLC gave the following results. In whole pancreas, GM3 represented 66.7% of total gangliosides detected; an asialoglycolipid comigrating with GM2, 2.0%; a ganglioside migrating between GM2 and GM1, 2.6%; GD3, 22.6%; and a GD1a-comigrating ganglioside, 6.1%. In isolated islets, these components were found at the following levels: GM3, 14.9%; GM2-comigrating glycolipid, 74.2%; a ganglioside migrating between GM2 and GM1, 9.8%; GD3, 1.1%; and the GD1a-comigrating ganglioside, not detectable.     

Presence of ATP-pyrophosphohydrolase in mouse pancreatic islets

The presence of an enzyme that hydrolyzes ATP to AMP and PPi was demonstrated in a 27,000 X g particulate and supernatant fraction of mouse pancreatic islets. The enzyme was stimulated by addition of Ca2+, Zn2+, and Co2+. Addition of calmodulin or trifluoperazine had no effect. In the presence of Ca2+ and Zn2+, the Michaelis constant (Km) for ATP was approximately 0.1 mM and the maximum velocity (Vmax) was close to 90 nmol X min-1 X mg protein-1. After preincubation of the islets for 30 min with 16.7 mM glucose or 5 mM glucose with 1 mM 3-isobutyl-1-methylxanthine (IBMX), a three- to fourfold increase in enzyme activity was seen. Direct addition of IBMX or cAMP to the enzyme assay also had a small stimulatory effect. Preincubation with the insulin secretagogues leucine and alpha-ketoisocaproic acid did not affect the enzyme activity. The possible function of the enzyme in pancreatic islets is discussed in relation to hypotheses given for the function of similar enzyme(s) in other tissues.     

Regulation of glucose metabolism in pancreatic islets

We evaluated the possible role of islet glucokinase in controlling the rate of islet glucose metabolism, and thereby the rate of glucose-induced insulin release. The activities of glucokinase, hexokinase, P-fructokinase, and glyceraldehyde-P dehydrogenase were quantitated in sonicated or isotonically homogenized islet preparations using pyridine nucleotide-dependent fluorometric assays. In sonicates, about 1/4 of the islet glucose phosphorylating activity was due to an enzyme with kinetic properties similar to glucokinase; 3/4 of the activity was due to hexokinase. The procedure for determining islet glucokinase activity was improved by centrifuging isotonic islet homogenates at 12,000 x g. The supernatant fraction was enriched for glucokinase. About 1/2 of the glucose phosphorylating activity in this fraction was due to glucokinase and 1/2 was due to hexokinase. The glucokinase activity in islet homogenates was !23 of the activity of hexokinase, 1/40 of the activity of P-fructokinase, and 1/400 of the activity of glyceraldehyde-P dehydrogenase. Detailed concentration dependency curves of glucose and mannose utilization were also obtained with intact isolated pancreatic rat islets. Glucose and mannose usage in islets was governed by two superimposed hyperbolic systems differing in Km and Vmax. A high Km system (Km for glucose 11 mM and for mannose 21 mM) predominated. A low Km system (Km for glucose 215 and for mannose 530 microM) contributed about 15% to the total activity. The available data with intact islets could be rationalized by the existence of two distinct hexose phosphorylating enzymes with differing capacities and kinetic properties. These enzymes, tentatively identified as glucokinase and hexokinase, could coexist in the same cell or could be distributed among different cell types. The possible physiologic significance of these results is discussed, emphasizing the idea of dual control of glycolysis and insulin release by glucokinase and hexokinase. An earlier proposal that glucokinase serves as glucoreceptor of beta-cells [J. Biol. Chem. 243:2730 (1968)] is greatly strengthened by the present studies.     

Tissue-engineered pancreatic islets: culturing rat islets in the chitosan sponge

Subcutaneous islet transplantation has become an attractive modality. With development of tissue-engineering techniques, it is possible to rectify the disadvantage of poor blood supply in the subcutaneous site by reconstruction of the capillary network. According to reports, the Chitosan sponge (CS) could be used for reconstruction of in vitro capillary-like network and could be used in artificial skin equivalent. In this study, we cultured the islets in CS for future application. CSs, having 200-500 microm pore size, were prepared by freeze-drying method. Rat islets were isolated from the pancreas of Lewis rats (10 weeks old, 280-300 g, male) by collagenase digestion followed by discontinuous dextran gradient centrifugation method. Each 20 islets were seeded equally into the CSs and were cultured for 62 days with various culture media such as RPMI-1640, Dulbecco's modified Eagle's medium (DMEM), and Eagle's MEM. They contained 10% fetal bovine serum (FBS) and 5 ml/L antibiotic-antimycotic mixed stock solution in the culture dishes. Insulin concentration both inside and outside of the islet-seeded CS was measured during culture. Changes in the morphology of islets were also observed in this study. Freshly isolated islets had a loose appearance with an irregular border, and most were seen as a single islet. Occasionally a cluster, consisting of 2-4 islets ranging mainly from 150 to 250 microm in diameter, was observed. Islets cultured in the CSs in different culture media retained initial morphology, which had well-delineated smooth borders for at least 53 days. The insulin release behavior of islets cultured in the CS showed constant secretory capacities for 49 days. After that they exhibited a rapid and definitive decline from the initial insulin release. Until this stage, insulin concentration in the CS was well maintained. The properties were dependent on culture medium used and insulin diffusion released from islets. This experiment is a new study model for establishment of islet culture in a three-dimensional matrix. Also extension of this observation will provide new insights for islet transplantation at the subcutaneous site by a tissue-engineering approach.     

Proliferation of transplanted allogeneic pancreatic islets

Recent studies showed enhanced regeneration of pancreatic islets in some circumstances. The purpose of our study was to investigate the proliferate potential of rat pancreatic islet cells in allogeneic grafts. Adult Lewis female rats and WAG male rats served as recipients and donors, respectively. Diabetes was induced by single intravenous (IV) injection of streptozotocin producing diabetes as confirmed by nonfasting plasma glucose >300 mg% on 3 consecutive days. Islet rejection was considered complete when glycemia exceeded 250 mg% and was confirmed by histopathological examination. To obtain long survival of allogeneic islets a tolerance-inducing method used allogeneic UV-B irradiated bone marrow transplantation into nonlethally selectively cytoreducted recipients with a donor-type splenocyte infusion followed by cyclophosphamide 200 mg/kg bw. Endocrine cell proliferation was assessed morphometrically using double immunostaining for pKi-67 and insulin or glucagon. Double immunolabelling, propidium iodide staining, and TUNEL assay were used to identify both proliferating and apoptotic cells. The rise of glycemia >350 mg/dL after graftectomy in euglycemic recipients was correlated with immunohistological examination, showing that the euglycemia was due to properly functioning pancreatic islet allotransplants. The immunohistochemical examination confirmed the presence of endocrine beta and alpha cells. In comparison with normal pancreas which showed 0.4 +/- 0.12%, pKi-67-positive cells, long-surviving grafts had a significantly higher proliferation capacity (5.61 +/- 0.94%; P <.001). In contrast, rejected grafts/control groups did not show significantly enhanced proliferation (0.73 +/- 0.19%), and had endocrine cells undergoing apoptosis. The incidence of apoptosis in endocrine cells within long-surviving graft appeared to be extremely low. In conclusion, the growth and death of endocrine cells in allogeneic grafts differ between accepted and rejected cases. The level of proliferation in the graft at day 150 was significantly higher compared with normal pancreatic beta cells.     

Size-dependent revascularization of transplanted pancreatic islets

For their survival and optimal function, pancreatic islets depend posttransplantation on a rapid and adequate revascularization. Native islets display a marked size-dependent heterogeneity in both angioarchitecture and degree of blood perfusion. This study evaluated whether there also are differences in the degree of revascularization of islets of different size when transplanted. Mouse pancreatic islets were isolated by collagenase digestion, and cultured in vitro for 4-7 days before transplantation. Groups of 200 islets with a diameter either exceeding or being below 100 microm were implanted beneath the left renal capsule of syngeneic C57 BL/6 mice. One month posttransplantation, graft-bearing kidneys were removed. Histological specimens were prepared and stained for endothelium with the lectin Bandeiraea simplicifolia. Pancreata from nontransplanted control animals were prepared similarly. The vascular density in transplanted islets was markedly lower than in native islets. However, islet transplants composed of small islets (<100 microm in diameter) had a vascular density in the endocrine tissue twice that in transplants of larger islets (>100 microm). The connective tissue stroma surrounding smaller islets was also more revascularized than in corresponding grafts with large islets. The vascular density in the connective tissue stroma surrounding the individual islets in the grafts was markedly higher than in the endocrine parts per se. These combined observations indicate that smaller islets have a higher capacity to stimulate regrowth of blood vessels following transplantation. Further studies on islet differences with regard to revascularization capacity may teach us strategies for treatment of transplanted islets to improve their revascularization.     

Isolation of viable human pancreatic islets

This article reports a method for the isolation of viable pancreatic islets from the human pancreas. Isolated islets were obtained from human pancreata of cadavers, patients undergoing surgical operations, and fetuses, using a freehand microdissection procedure. Viability was assessed by light microscopy of sections stained with aldehyde fuchsin and by measuring the insulin output of islets in response to a glucose stimulus in vitro using a perifusion system. Ten pieces of cadaver pancreas were studied. Islets were isolated from 6 specimens and in 5 of these were shown to respond to a glucose stimulus in vitro. Histologically the islets showed minimal damage with slight degranulation of the beta cells. Five pieces of pancreas removed at operation were studied as well. Islets were isolated in all cases, but only 2 showed a response to a glucose stimulus. Pancreata from a 26-week and 34-week human fetus were also studied. It was not possible to microdissect islets from either case, but small pieces of pancreas from the 26-week fetus were shown to respond to a glucose stimulus by producing a significant increase in insulin output.

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Résumé Cet article décrit une méthode pour isoler des ilÔts de Langérhans viables à partir de pancréas humain. Une technique de microdissection manuelle a permis d'isoler ces ilÔts à partir de pancréas humains obtenus chez des patients opérés, chez des donneurs cadavériques et chez des foetus. Leur viabilité a été établie par l'examen en microscopie optique de sections tissulaires préparés à la fuchsine aldehyde et par mesure de la sécrétion d'insuline en réponse à une stimulation au glucose au moyen d'un système de périfusion in vitro. Dix échantillons de pancréas cadavériques ont été étudiés. On a réussi à isoler des ilÔts chez six d'entre eux et cinq ont répondu au test de stimulation au glucose in vitro. à l'examen histologique, les ilÔts ne présentaient que peu de modification et une légère dégranulation des cellules bÊta. Cinq spécimens de pancréas obtenus chirurgiculement ont été étudiés. On a pu isoler des ilÔts dans tous les cas, mais seulement deux ont répondu à la stimulation au glucose. Finalement on a aussi étudié des échantillons pancréatiques obtenus chez deux foetus humains de 24 et 36 semaines respectivement. Chez ni l'un ni l'autre il n'a été possible d'isoler des ilÔts, mais de petites tranches de tissus pancréatiques préparés chez le foetus de 26 semaines et soumis au test de stimulation au glucose ont augmenté significativement leur sécrétion d'insuline.

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Part of the cost of this project was defrayed by a grant from the Wellcome Trust.     

Nonviral transfection of intact pancreatic islets

Ex vivo gene transfer offers a potential means to introduce genes into cells, which may play an important role in preventing graft rejection and inducing graft tolerance. This study examined the efficiency and toxicity of several lipid-based transfection reagents (LipofectAMINE, DOTAP, and DOSPER) in intact pancreatic islets. Isolated islets were transfected with a pCMV-beta-galactosidase plasmid using several DNA/liposome ratios (1:12) of liposomes (3-72 microl) and DNA (3 and 6 microg). Transfection efficiency was quantified by microscopic evaluation of beta-galactosidase gene expression in whole intact islets. Functionality of the transfected islets was measured by insulin response to glucose solutions. All transfection reagents evaluated in this study transfected cells within the islets. As expected, untransfected controls and transfected islets with DNA alone did not express beta-gal. The highest transfection efficiency and functional viability were obtained following a 48-h incubation after exposure to the transfection mixtures as follows: 3 microl DNA and 18 microl DOTAP/ml (1:6 ratio), 6 microg DNA and 12 microl DOSPER/ml (1:2 ratio), or 6 microg DNA and 12 microl Lipofect-AMINE/ml (1:2 ratio). The highest rate of transfected cells per islet was obtained using DOTAP. In vitro functionality was not significantly different between DOTAP and nontreated controls. However, optimal transfection efficiency doses of LipofectAMINE and DOSPER significantly reduced the stimulated insulin response of the transfected islets (p < 0.05, ANOVA). The calculated stimulation index (SI) was 7.8+/-0.6 (mean +/- SEM) for DOTAP-transfected islets compared with 8.4+/-0.5 for nontransfected control islets (p = ns). The SI of DOSPER- and LipofectAMINE-transfected islets was significantly lower (6.1+/-0.5 and 3.4+/-0.5, respectively, p < 0.05). Lipid-based transfection using DOTAP at a DNA/lipid ratio of 1:6 provides an effective means of ex vivo gene delivery without compromising in vitro functionality of the transfected islets.     

Regulation of xenogeneic porcine pancreatic islets

The use of xenogeneic porcine pancreatic islets has been shown to be a potentially promising alternative to using human allogeneic islets to treat insulin-dependent type 1 diabetes (T1D). This article provides an overview of the existing FDA regulatory framework that would be applied to the regulation of clinical trials utilizing xenogeneic porcine pancreatic islets to treat T1D.     

Regulation of ATP/ADP in pancreatic islets

ATP and ADP levels are critical regulators of glucose-stimulated insulin secretion. In many aerobic cell types, the phosphorylation potential (ATP/ADP/P(i)) is controlled by sensing mechanisms inherent in mitochondrial metabolism that feed back and induce compensatory changes in electron transport. To determine whether such regulation may contribute to stimulus-secretion coupling in islet cells, we used a recently developed flow culture system to continuously and noninvasively measure cytochrome c redox state and oxygen consumption as indexes of electron transport in perifused isolated rat islets. Increasing substrate availability by increasing glucose increased cytochrome c reduction and oxygen consumption, whereas increasing metabolic demand with glibenclamide increased oxygen consumption but not cytochrome c reduction. The data were analyzed using a kinetic model of the dual control of electron transport and oxygen consumption by substrate availability and energy demand, and ATP/ADP/P(i) was estimated as a function of time. ATP/ADP/P(i) increased in response to glucose and decreased in response to glibenclamide, consistent with what is known about the effects of these agents on energy state. Therefore, a simple model representing the hypothesized role of mitochondrial coupling in governing phosphorylation potential correctly predicted the directional changes in ATP/ADP/P(i). Thus, the data support the notion that mitochondrial-coupling mechanisms, by virtue of their role in establishing ATP and ADP levels, may play a role in mediating nutrient-stimulated insulin secretion. Our results also offer a new method for continuous noninvasive measures of islet cell phosphorylation potential, a critical metabolic variable that controls insulin secretion by ATP-sensitive K(+)-dependent and -independent mechanisms.     

Intracerebral allotransplantation of purified pancreatic endocrine cells and pancreatic islets in diabetic rats

Allogeneic pancreatic endocrine cells (PEC) and whole islets from inbred Lewis (AgB 1/1) and outbred Wistar rats were implanted intracerebrally (i.c.) into two designated areas of streptozotocin-induced diabetic ACI (AgB 4/4) rats across the major histocompatibility barrier. All the transplants of PEC from Lewis (n = 12) and Wistar (n = 7) donors remained functional for an observation period in excess of 200 days. In contrast, only 3/6 Lewis and 3/9 Wistar whole-islet transplants were able to maintain function for a prolonged period. Recipients with functional PEC or islet allografts had normalized nonfasting blood glucose (BG) in the 24-hr. BG profile, and they maintained a steady body weight gain. ACI recipients of PEC from Lewis rats had glucose disappearance K rates of 1.3 +/- 0.3 (mean +/- SE) and a normal basal BG level in 4 hr following the i.v. glucose load. Histological section of the brain tissues with successful i.c. islet or PEC grafts up to a duration of 5 1/2 months revealed healthy endocrine cells in the cortex and the subarachnoid space. These grafts were permeated with capillaries but devoid of exocrine tissues or lymphoid cell infiltration. These observations suggest that the brain is an immunologically privileged site, and that it is a hospitable site for the pancreatic endocrine cell suspension. However, the immunological protection offered to allogeneic transplants by the brain is incomplete, and purified PEC must be employed to ensure consistent long-term allograft survival.     

微囊新生猪胰岛细胞体外培养的研究

用胶原酶消化和体外培养的方法成功地获得了大量有活性和较纯的新生猪胰岛细胞，用微囊包膜技术包裹胰岛细胞，并进行培养。由放射免疫药盒对培养液中胰岛素的含量测定，胰岛素释放试验和组织学检查等体外实验所得的数据，证实了微囊和非微囊的新生猪胰岛的活性和分泌功能的差异不显著，说明本包膜材料和一步法制囊新技术对胰岛细胞无损伤，微囊包膜后胰岛细胞的活性和分泌功能良好。     

Glucose-induced accumulation of fructose-2,6-bisphosphate in pancreatic islets

Rat islets contain the acid-labile activator of phosphofructokinase, fructose-2,6-bisphosphate. The islet content in activator is higher in islets exposed to glucose (16.7 mM) than in islets deprived of glucose. The islets display fructose-6-phosphate, 2-kinase activity with a Km for fructose-6-phosphate close to 0.08 mM. Glucose fails to affect the activity of this enzyme. It is proposed that the effect of glucose to increase the islet content of fructose-2,6-bisphosphate is attributable, in part at least, to the glucose-induced increase in the concentration of fructose-6-phosphate in the islet cells.     

Interactions of cholecystokinin and glucose in rat pancreatic islets

The effects of sulfated cholecystokinin (CCK-8S) and glucose on insulin secretion and polyphosphoinositide (PPI) metabolism were studied in isolated rat islets. Both agonists stimulate PPI hydrolysis, inositol phosphate accumulation, 3H efflux from [3H]inositol-prelabeled tissue, and 45Ca efflux from prelabeled cells. However, the effects of CCK-8S on PPI metabolism are considerably greater than those of glucose. Furthermore, the effects of CCK-8S on PPI and Ca2+ metabolism are observed whether islets are incubated in either 2.75 or 7 mM glucose, but CCK-8S only stimulates insulin secretion (a biphasic response) when the higher glucose concentration is present. Addition of 1 microM forskolin to islets incubated in media containing 2.75 mM glucose does not influence basal insulin secretion but sensitizes the islets to the action of CCK-8S. In the presence of forskolin, CCK-8S induces a very marked first phase but no second phase of insulin secretion. We postulate that CCK-8S acts in this tissue via receptor-linked PPI hydrolysis, leading to an inositol trisphosphate-induced Ca2+ efflux. These receptor-mediated effects of CCK-8S are not altered either by the ambient glucose concentration or the cAMP content of the islets, but these two factors determine the responsiveness of the islets (in terms of insulin secretion) to a given CCK-8S signal.     

Immunoisolation strategies for the transplantation of pancreatic islets

Several different types of systems employing selectively permeable membranes and matrix supports for cells have been successfully tested in animals. Results in diabetic animals indicate that these systems can function for periods of several months to several years without the use of any of immunosuppression. This approach has the potential not only to allow the transplantation of islets across wide species barriers but that it can be achieved using injectable microreactors fabricated from biodegradable polymers. A new prototype of minimal volume alginate/polyaminoacidic microcapsules which retain immunoisolatory, biocompatibility and functional properties that seem to match those of conventional-size microcapsules was developed. Since coherent microcapsules tightly envelop each islet, any redundant space between membrane and islet is virtually eliminated. Consequently, these microcapsules occupy an extremely this space, thus addressing a major problem associated with conventional size microspheres.