Genetic engineering of a suboptimal islet graft with A20 preserves beta cell mass and function

Transplantation of an excessive number of islets of Langerhans (two to four pancreata per recipient) into patients with type I diabetes is required to restore euglycemia. Hypoxia, nutrient deprivation, local inflammation, and the beta cell inflammatory response (up-regulation of NF-kappaB-dependent genes such as inos) result in beta cell destruction in the early post-transplantation period. Genetic engineering of islets with anti-inflammatory and antiapoptotic genes may prevent beta cell loss and primary nonfunction. We have shown in vitro that A20 inhibits NF-kappaB activation in islets and protects from cytokine- and death receptor-mediated apoptosis. In vivo, protection of newly transplanted islets would reduce the number of islets required for successful transplantation. Transplantation of 500 B6/AF(1) mouse islets into syngeneic, diabetic recipients resulted in a cure rate of 100% within 5 days. Transplantation of 250 islets resulted in a cure rate of only 20%. Transplantation of 250 islets overexpressing A20 resulted in a cure rate of 75% with a mean time to cure of 5.2 days, comparable to that achieved with 500 islets. A20-expressing islets preserve functional beta cell mass and are protected from cell death. These data demonstrate that A20 is an ideal cytoprotective gene therapy candidate for islet transplantation.     

Isolation,transplantation, and functional studies of adult porcine islets of Langerhans

Transplantation of islets of Langerhans is a possible treatment for type-I diabetes mellitus. However, there is a shortage of donors for such transplantations and the pig may be an alternative source of donor organs. The aims of the study reported here were to establish a method for adult porcine islet isolation that was based on enzymatic digestion using Liberase PI in a semiautomatic set-up, and to evaluate the in vitro and in vivo function of isolated islets. After overnight culture, isolated islets, from five of seven batches, had poor insulin response to an in vitro glucose challenge that was only partially increased by additional challenge with arginine. More than 50% of DNA and 90% of the insulin content was lost during a one-week culture period. With some batch-to-batch variation, in 15 of 25 cases, 4,000 to 7,000 porcine islets cured streptozotocin diabetic nude mice within three weeks following transplantation. In conclusion, it is possible to isolate viable islets from adult pigs, using a semiautomatic set-up. With batch-to-batch variation, the islets are able to revert diabetes mellitus when transplanted to diabetic nude mice.     

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes ^1,2. More recently, advances in human islet transplantation have further strengthened this view ^1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.     

Overexpression of suppressor of cytokine signaling 1 in islet grafts results in anti-apoptotic effects and prolongs graft survival

AimsA significant portion of islet grafts are destroyed by apoptosis and fail to become functional after transplantation. Strategies that enhance islet resistance to apoptosis may prevent graft loss. The aim of this study was to investigate whether overexpression of suppressor of cytokine signaling 1 (SOCS1) in islet grafts could achieve an anti-apoptotic effect and prolong graft survival.Main methodsWe used a chimeric adenovirus vector (Ad5F35) to enhance SOCS1 expression in isolated rat islets, and assessed its protective action against TNF-α-induced apoptosis. After transplanting SOCS1-overexpressing islets into allogeneic recipients with streptozotocin-induced diabetes, graft survival and in situ apoptosis were analyzed using immunohistochemistry.Key findingsThe isolated rat islets infected with Ad5F35–SOCS1 showed significantly higher SOCS1 expression than Ad5F35–EGFP and mock infected islets. The Ad5F35 transfection and SOCS1 overexpression on islets did not affect their insulin secretory function. After treatment with rat TNF-α and cycloheximide in vitro, Ad5F35–-SOCS1 infected islets exhibited a lower apoptotic ratio than controls (Ad5F35–EGFP and mock infected islets). The diabetic recipients transplanted with Ad5F35–SOCS1 infected islets displayed longer time of normoglycemia than recipients transplanted with mock infected islets. Furthermore, histological analysis indicated that the infected grafts with local overexpression of SOCS1 showed decreased apoptosis in the early post-transplant period.SignificanceThese results demonstrate that overexpression of SOCS1 in islet grafts prior to transplantation can significantly protect them from apoptotic loss and prolong their survival. This approach might find a clinical counterpart.     

Transplantation and in vitro perifusion of rat islets of Langerhans after slow cooling and warming in the presence of either glycerol or dimethyl sulfoxide

The cryoprotectants dimethyl sulfoxide (Me2SO) and glycerol have been used for the cryopreservation of fetal rat pancreases but only Me2SO has been reported for the cryopreservation of adult rat islets. Since glycerol may be preferred to Me2SO for clinical use, this study was undertaken to compare the effectiveness of these cryoprotectants during the slow cooling of isolated adult rat islets. Islets of Langerhans prepared from the pancreases of WAG rats by collagenase digestion were stored at -196 degrees C after slow cooling (0.3 degrees C/min) to -70 degrees C in the presence of multimolar concentrations of either Me2SO or glycerol. Samples were rewarmed slowly (approximately 10 degrees C/min) and dilution of the cryoprotectant was achieved using medium containing sucrose. Function was assessed by determination of the time course of the glucose-induced insulin release during in vitro perifusion at 37 degrees C and also by isograft transplantation. Transplants were carried out by intraportal injection of a minimum of 1700 frozen and thawed islets into streptozotocin-induced diabetic recipients and tissue function was assessed by monitoring blood glucose levels and body weight changes. Without exception the islets frozen and thawed in the presence of glycerol failed to reduce high serum glucose levels of recipient rats and in vitro dynamic release curves showed to demonstrate a glucose-sensitive insulin release pattern. Reversal of the diabetic conditions was achieved in two of five animals receiving islets which had been frozen and thawed with 2 M Me2SO; and in one of three animals receiving islets cryopreserved with 3 M Me2SO. Nevertheless, perifusion studies showed that the pattern of insulin secretion from groups of cryopreserved islets which did show an ability to secrete insulin was atypical compared with that of untreated controls, suggesting that the tissue was altered or damaged in some way.     

Improved function of rat islets upon co-microencapsulation with Sertoli's cells in alginate/poly-L-ornithine

The purpose of this study was to assess whether Sertoli's cells would improve functional performance of homologous pancreatic islets within microcapsules. Purified rat Sertoli's cells were co-enveloped with islets in microcapsules that had been fabricated with alginic acid and poly-L-ornithine. Confocal laser microscopy was used to determine any mitogenic effects of Sertoli's cells on islets beta-cells. Insulin secretion from islets, with or without Sertoli's cells, was examined, and grafts of Sertoli's cells with islets in microcapsules into diabetic mice were carried out. Co-incubation of Sertoli's cells with islets resulted in a significant increase in the islet beta-cell mitotic rate, which was coupled with significantly higher insulin release under glucose stimulation, as compared to controls. Grafts of co-microencapsulated Sertoli's cells with islets resulted in prolongation of the achieved normoglycemia in the animals receiving Sertoli's cells with islets as compared to controls that received islets only. Sertoli's cells do promote mitogenic activities upon in vitro co-incubation with islets, whose in vitro functional and in vivo post-transplant consequences were evident. Sertoli's cells could, therefore, be co-microencapsulated with islets for transplantation in diabetic recipients.     

Islet transplantation in experimental diabetes in the rat. III. Studies in allogeneic streptozotocin-treated rats.

Diabetes of various degrees of severity was induced experimentally in rats by different doses of streptozotocin. These animals served as recipients for isolated islets of Langerhans from allogeneic donors. The islets were transplanted to different regions in the organism by paravascular or intravascular injection. As in pancreatectomized rats, the endocrine effect of the islets was only transient and consisted of disappearance of glycosuria, normalization of blood glucose and amelioration of intravenous glucose tolerance tests. When the islets were injected intravascularly (lung, liver) the influence of the transplanted islets was observable over a longer period than after subcutaneous or another paravascular transplantation. As in pancreatectomized animals, the period of survival was markedly prolonged in rats which had received a transplant compared to those which had not. The islets responded to glucose stimulation in vivo with insulin secretion similar to that of control rats, while only a very slight elevation of the low basis levels in streptozotocin-treated rats was observed.     

Pancreatic islets engineered with SA-FasL protein establish robust localized tolerance by inducing regulatory T cells in mice

Allogeneic islet transplantation is an important therapeutic approach for the treatment of type 1 diabetes. Clinical application of this approach, however, is severely curtailed by allograft rejection primarily initiated by pathogenic effector T cells regardless of chronic use of immunosuppression. Given the role of Fas-mediated signaling in regulating effector T cell responses, we tested if pancreatic islets can be engineered ex vivo to display on their surface an apoptotic form of Fas ligand protein chimeric with streptavidin (SA-FasL) and whether such engineered islets induce tolerance in allogeneic hosts. Islets were modified with biotin following efficient engineering with SA-FasL protein that persisted on the surface of islets for >1 wk in vitro. SA-FasL-engineered islet grafts established euglycemia in chemically diabetic syngeneic mice indefinitely, demonstrating functionality and lack of acute toxicity. Most importantly, the transplantation of SA-FasL-engineered BALB/c islet grafts in conjunction with a short course of rapamycin treatment resulted in robust localized tolerance in 100% of C57BL/6 recipients. Tolerance was initiated and maintained by CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells, as their depletion early during tolerance induction or late after established tolerance resulted in prompt graft rejection. Furthermore, Treg cells sorted from graft-draining lymph nodes, but not spleen, of long-term graft recipients prevented the rejection of unmodified allogeneic islets in an adoptive transfer model, further confirming the Treg role in established tolerance. Engineering islets ex vivo in a rapid and efficient manner to display on their surface immunomodulatory proteins represents a novel, safe, and clinically applicable approach with important implications for the treatment of type 1 diabetes.     

Low‐adhesive ethylene vinyl alcohol–based packaging to xenogeneic islet encapsulation for type 1 diabetes treatment

Transplantation of encapsulated porcine islets is proposed to treat type 1 diabetes. However, the envelopment of fibrous tissue and the infiltration of immune cells impair islet function and eventually cause implant failure. It is known that hemodialysis using an ethylene vinyl alcohol (EVOH) membrane results in minor tissue responses. Therefore, we hypothesized that using a low‐adhesive EVOH membrane for encapsulation may prevent host cell accumulation and fibrous capsule formation. In this study, rat islets suspended in chitosan gel were encapsulated in bags made from highly porous EVOH membranes, and their in vitro insulin secretion function as well as in vivo performance was evaluated. The results showed that the EVOH bag did not affect islet survival or glucose‐stimulated insulin secretion. Whereas naked islets were dysfunctional after 7 days of culture in vitro, islets within the EVOH bag produced insulin continuously for 30 days. Streptozotocin‐induced diabetic mice were given islets–chitosan gel–EVOH implants intraperitoneally (650–800 islets equivalent) and exhibited lower blood glucose levels and regained body weight during a 4‐week observation period. The transplanted mice had higher levels of serum insulin and C‐peptide, with an improved blood glucose disappearance rate. Retrieved implants had minor tissue adhesion, and histology showed a limited number of mononuclear cells and fibroblasts surrounding the implants. No invasion of host cells into the EVOH bags was noticed, and the encapsulated islets were intact and positive for insulin–glucagon immunostaining. In conclusion, an EVOH bag can protect encapsulated islets, limit fibrous capsule formation, and extend graft function.     

Cold storage preservation of islet and pancreas grafts as assessed by in vivo function after transplantation to diabetic hosts

The major goal of hypothermic (4–8 °C) preservation of intact pancreases or isolated islets will be to provide sufficient time for HLA typing, cross matching, selection, and preparation of recipients—logistical efforts requiring 12–72 hr for clinical kidney transplantation, usually <48 hours. Some investigators have studied in vitro function of islets after cold storage, but the critical test of viability—permanent restoration of normoglycemia after transplantation to diabetic recipients—has been tested in only a few experiments. Reversal of hyperglycemia by syngeneic or autogenic transplants in diabetic animals has been achieved after CS of dispersed pancreatic tissue from neonatal rats in GIB media for ? 146 hr, adult dogs in TCM 199 for ?24 hr, and adult DL-ethionine-treated rats in RPMI 1640 for ?72 hr. In the neonatal rat donor model, intravenous glucose tolerance test (IVGTT) results were similar in recipients of fresh or stored islets; in the dog model, IVGTT test results were variable, but generally inferior in recipients of stored as compared to fresh islets; in the adult rat donor model, recipients of ?24-hr coldstorage islets had insulin and IVGTT K values similar to those of recipients of fresh islets, but the success rate progressively declined for CS times >24 hr. Various agents were added to the media, but the need or the optimal concentrations were not critically determined by using different recipes for different groups of recipients. Cold storage of intact pancreas autografts has been tested in dogs; simple electrolyte solutions are satisfactory for 24 hr, but only a silica gel-filtered plasma-based solution has been reliable for 48 hr. Pulsatile machine perfusion (PMP) of canine pancreas grafts for 24 hr has had a success rate similar to CS in some experiments and lower in others. PMP has been almost totally unreliable for >24 hr. Further refinements are needed if preservation of islets for >24 hr and pancreases for >48 hr are to be consistently successful. If current experimental techniques are effective for human islets or pancreases, however, these times are sufficient to complete the logistical maneuvers required before transplantation.     

Islet cell transplantation

Islet cell transplantation is an attractive alternative therapy to conventional insulin treatment or vascularized whole pancreas transplantation for type 1 diabetic patients. It represents a successful example of somatic cell therapy in humans based on complex procedures for islet isolation from whole pancreas. The islets, that are only 1% of the total pancreas tissue, are isolated by two steps method starting with collagenase digestion that operates a rapid dissociation of the stromal component of the gland, while preserving islet anatomical integrity. After digestion, islets are then separated from exocrine tissue by centrifugation in density gradients. Transplantation consists of a simple injection of few milliliter-purified tissue in the portal vein through a percutaneous trans-hepatic approach performed in local anesthesia. Several studies have now demonstrated that islet transplant can replace pancreatic endocrine function without major side effects and with liver viability preservation in selected patients affected by long-term type 1 diabetes. It can restore endogenous insulin secretion, achieve insulin independence in more than 80% of patients, and recover the metabolism of glucose, protein and lipids. Improved control of glycated HbA1c, reduced risk of recurrent hypoglycemia and of diabetic complications are also seen as important benefits of islet cell transplantation, irrespective of the status of insulin independence. Many protocols are now on going for reduction of immunosuppression therapy in recipients, induction of tolerance, and prolongation of graft function.     

Mitochondrial metabolism reveals a functional architecture in intact islets of Langerhans from normal and diabetic Psammomys obesus

The cells within the intact islet of Langerhans function as a metabolic syncytium, secreting insulin in a coordinated and oscillatory manner in response to external fuel. With increased glucose, the oscillatory amplitude is enhanced, leading to the hypothesis that cells within the islet are secreting with greater synchronization. Consequently, non-insulin-dependent diabetes mellitus (NIDDM; type 2 diabetes)-induced irregularities in insulin secretion oscillations may be attributed to decreased intercellular coordination. The purpose of the present study was to determine whether the degree of metabolic coordination within the intact islet was enhanced by increased glucose and compromised by NIDDM. Experiments were performed with isolated islets from normal and diabetic Psammomys obesus. Using confocal microscopy and the mitochondrial potentiometric dye rhodamine 123, we measured mitochondrial membrane potential oscillations in individual cells within intact islets. When mitochondrial membrane potential was averaged from all the cells in a single islet, the resultant waveform demonstrated clear sinusoidal oscillations. Cells within islets were heterogeneous in terms of cellular synchronicity (similarity in phase and period), sinusoidal regularity, and frequency of oscillation. Cells within normal islets oscillated with greater synchronicity compared with cells within diabetic islets. The range of oscillatory frequencies was unchanged by glucose or diabetes. Cells within diabetic (but not normal) islets increased oscillatory regularity in response to glucose. These data support the hypothesis that glucose enhances metabolic coupling in normal islets and that the dampening of oscillatory insulin secretion in NIDDM may result from disrupted metabolic coupling.     

Anti-caspase-3 preconditioning increases proinsulin secretion and deteriorates posttransplant function of isolated human islets

Human islet isolation is associated with adverse conditions inducing apoptosis and necrosis. The aim of the present study was to assess whether antiapoptotic preconditioning can improve in vitro and posttransplant function of isolated human islets. A dose-finding study demonstrated that 200 μmol/L of the caspase-3 inhibitor Ac-DEVD-CMK was most efficient to reduce the expression of activated caspase-3 in isolated human islets exposed to severe heat shock. Ac-DEVD-CMK-pretreated or sham-treated islets were transplanted into immunocompetent or immunodeficient diabetic mice and subjected to static glucose incubation to measure insulin and proinsulin secretion. Antiapoptotic pretreatment significantly deteriorated graft function resulting in elevated nonfasting serum glucose when compared to sham-treated islets transplanted into diabetic nude mice (p < 0.01) and into immunocompetent mice (p < 0.05). Ac-DEVD-CMK pretreatment did not significantly change basal and glucose-stimulated insulin release compared to sham-treated human islets but increased the proinsulin release at high glucose concentrations (20 mM) thus reducing the insulin-to-proinsulin ratio in preconditioned islets (p < 0.05). This study demonstrates that the caspase-3 inhibitor Ac-DEVD-CMK interferes with proinsulin conversion in preconditioned islets reducing their potency to cure diabetic mice. The mechanism behind this phenomenon is unclear so far but may be related to the ketone CMK linked to the Ac-DEVD molecule. Further studies are required to identify biocompatible caspase inhibitors suitable for islet preconditioning.     

Islet transplantation in experimental diabetes of the rat. X. Induction of cytotoxic antibodies and islet-cell-surface-antibodies after homologous islet transplantation in rats

The mechanisms of islet allograft rejection are still unknown. 350 allogeneic rat islets (BDE/Han) were transplanted into the liver of Streptozotocin-diabetic rats (Lewis/Han). Animals were killed either on day 4, 7, 10 or 14. Immunofluorescence analysis for islet-cell-surface-antibodies (ICSA) was performed on BDE-islet-single-cells yielded by Collagenase-Dispase digestion. In order to detect cytoplasmic islet cell antibodies, immunofluorescence technique was used on BDE-pancreas cryosections. Cytotoxicity assay was tried out using 51Cr-release-test. After successful intraportal transplantation, rejection took place between day 7 and 10. ICSA could be observed on day 10 and 14. Cytoplasmic antibodies were not detected at any time. A specific cytotoxic activity in the recipient serum revealed itself on day 10 (23.0%) and day 14 (27.7%). Transplantation of freshly isolated allogeneic islets induced ICSA and cytotoxic antibodies. These results indicate an involvement of humoral mechanisms in islet allograft rejection.     

Visualizing superoxide production in normal and diabetic rat islets of Langerhans

Oxygen free radicals have been implicated in beta-cell dysfunction and apoptosis associated with type 1 and type 2 diabetes mellitus. The roles of free radicals in diabetes have thus far been defined indirectly by monitoring oxidative tissue damage and the effects of antioxidants, free radical scavengers, and overexpression of superoxide dismutase. We employed the superoxide-mediated oxidation of hydroethidine to ethidium to dynamically and directly assess the relative rates of mitochondrial superoxide anion generation in isolated islets in response to glucose stimulation. Superoxide content of isolated islets increased in response to glucose stimulation. We next compared the oxyradical levels in Zucker lean control and Zucker diabetic fatty rat islets by digital imaging microfluorometry. The superoxide content of Zucker diabetic fatty islets was significantly higher than Zucker lean control islets under resting conditions, relatively insensitive to elevated glucose concentrations, and correlated temporally with a decrease in glucose-induced hyperpolarization of the mitochondrial membrane. Importantly, superoxide levels were elevated in islets from young, pre-diabetic Zucker diabetic fatty animals. Overproduction of superoxide was associated with perturbed mitochondrial morphology and may contribute to abnormal glucose signaling found in the Zucker diabetic fatty model of type 2 diabetes mellitus.     

Prolonged local expression of anti-CD4 antibody by adenovirally transduced allografts can promote long-term graft survival

BACKGROUND: Currently, successful transplantation of allografts requires the systemic use of immunosuppressive drugs. These can cause serious morbidity due to toxicity and increased susceptibility to cancer and infections. Local production of immunosuppressive molecules limited to the graft site would reduce the need for conventional, generalized immunosuppressive therapies and thus educe fewer side effects. This is particularly salient in a disease like type 1 diabetes, which is not immediately life-threatening yet islet allografts can effect a cure. METHODS: We studied the efficacy of locally produced anti-CD4 antibody, mediated by adenovirus (Adv-anti-CD4) transduction of islets, to enhance allograft survival. Adenovirus-transduced islets were transplanted under the kidney capsule of diabetic recipients and graft rejection determined by monitoring blood glucose levels. RESULTS: Adv-anti-CD4 transduction of mouse islets afforded protection against allogeneic rejection after transplantation into fully mismatched recipients. In some recipients, the islet allograft survival was prolonged (persisting for at least 15 weeks), corresponding to the prolonged expression of the anti-CD4 antibody. The effect was local, as absence of CD4+ T lymphocytes was observed primarily at the graft site. CONCLUSIONS: Immunosuppressive effects can be restricted locally by our strategy. Local production of a single antibody against one subset of T lymphocytes can protect mouse islets from allograft rejection during transplantation to treat diabetes. Our findings foreshadow that this strategy may be even more effective when a combination of antibodies are used and that similar strategies may prevent xenograft rejection.     

Encapsulation of individual pancreatic islets by sol-gel SiO2: a novel procedure for perspective cellular grafts

Pancreatic rat islets are encapsulated by a siliceous layer deposited on the surface of single islets upon reaction with gaseous siliceous precursors. The process preserves original islet dimensions and does not suppress viability or function. The encapsulated material is homogeneously distributed on the islet surface, and layer thickness can be controlled in the 0.1–2.0 μm interval. Dynamic perfusion experiments with glucose stimulation were carried out in both encapsulated and non-encapsulated islets. Results were treated according to a kinetic model presented here for the analysis of perfusion data; the model tested by literature data, was used to substantiate the diffusion features of the siliceous layer, which does not affect mass transfer of insulin but which modifies the texture of the islet surface tissue. The clinical potential of silica encapsulation was demonstrated by in vivo experiments using encapsulated islets transplanted into diabetic rats. Transplantation was carried out in both inbred and outbred rats and indicated prolonged restoration of normal glycaemia levels and protection from immunological attack.     

In vitro and in vivo protective effect of Ganoderma lucidum polysaccharides on alloxan-induced pancreatic islets damage

This study was undertaken to investigate the protective effect against alloxan-induced pancreatic islets damage by Ganoderma lucidum Polysaccharides (Gl-PS) isolated from the fruiting body of Ganoderma lucidum (Leyss. ex Fr.) Karst. In vitro, alloxan caused dose-dependent toxicity on the isolated pancreatic islets. Pre-treatment of islets with Gl-PS for 12 h and 24 h significantly reversed alloxan-induced islets viability loss. Gl-PS was also found to inhibit the free radicals production induced by alloxan in the isolated pancreatic islets using confocal microscopy. Gl-PS dose-dependently increased serum insulin and reduced serum glucose levels when pretreated intragastrically for 10 days in alloxan-induced diabetic mice. It was found that the pancreas homogenates had higher lipid peroxidation products in alloxan-treated mice than in the Gl-PS-treated animals. Aldehyde fuchsin staining revealed that alloxan caused nearly all the beta cells disappearing from the pancreatic islets, while Gl-PS partly protected the beta cells from necrosis. Alloxan (60 mg/kg) induced NF-kappa B activation in the pancreas at 30 min after injection, pretreatment with Gl-PS inhibited alloxan-induced activation of NF-kappa B. These results suggest that Gl-PS was useful in protecting against alloxan-induced pancreatic islets damage in vitro and in vivo; one of the mechanisms is through its scavenging ability to protect the pancreatic islets from free radicals-damage induced by alloxan.     

Ubiquitination is involved in glucose-mediated downregulation of GIP receptors in islets

Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal hormone that has a potent stimulatory effect on insulin release under conditions of normal glucose tolerance. However, its insulinotropic effect is reduced or even absent entirely in type 2 diabetic patients. In this study, we addressed the role of glucose concentration in the diabetic range of >or=11 mM, i.e., hyperglycemia per se, as a cause of the lack of response to GIP. Culturing rat and human pancreatic islets in >or=11 mM glucose for up to 24 h resulted in prevention of GIP-mediated intracellular cAMP increase compared with culturing in 5 mM glucose. Western blot analysis revealed a selective 67 +/- 2% (rat) and 60 +/- 8% (human) decrease of GIP-R expression in islets exposed to >or=11 mM glucose compared with 5 mM glucose (P < 0.001). We further immunoprecipitated GIP-R from islets and found that GIP-R was targeted for ubiquitination in a glucose- and time-dependent manner. Downregulation of GIP-R was rescued by treating isolated islets with proteasomal inhibitors lactacystin and MG-132, and the islets were once again capable of increasing intracellular cAMP levels in response to GIP. These results suggest that the GIP-R is ubiquitated, resulting in downregulation of the actions of GIP.