Magnitude and mechanisms of glucose counterregulation following islet transplantation in patients with type 1 diabetes suffering from severe hypoglycaemic episodes.

AIMS/HYPOTHESIS: Pancreatic islet transplantation stabilises glycaemic control in type 1 diabetes mellitus patients with neuroglycopoenia, despite them not achieving insulin independence because of limited graft function. However, the extent and underlying metabolic pathways of restored glucose counterregulation are unknown. We therefore compared systemic glucose turnover, including lactate gluconeogenesis (GN) and muscle glucose uptake, in individuals with type 1 diabetes who were transplant recipients with partial graft function (T1DM/ITx(+)), matched non-transplanted individuals with type 1 diabetes (T1DM/ITx(-)) and matched healthy non-diabetic individuals. METHODS: Participants (n = 12 in each group) underwent a euglycaemic and a hypoglycaemic (2.5-2.8 mmol/l) hyperinsulinaemic clamp (0.8 mU kg(-1) min(-1)) in a randomised crossover fashion. Systemic and skeletal muscle glucose and lactate kinetics were assessed using a combination of isotopic and forearm balance techniques. RESULTS: Whole-body glucose counterregulation, the difference in glucose infusion rates required to maintain the glycaemic goal between the hypoglycaemic and euglycaemic clamps, was improved in T1DM/ITx(+) (7.8 ± 1.3 µmol kg(-1) min(-1)) compared with T1DM/ITx(-) (0.3 ± 0.9 µmol kg(-1) min(-1)), but was ~45% lower than in controls (14.1 ± 2.1 µmol kg(-1) min(-1)). Increased endogenous glucose production (EGP) and decreased systemic glucose disposal accounted for 49% and 39% of glucose counterregulation in T1DM/ITx(+), respectively, compared with 60% and 36% in controls. Lactate GN increased in T1DM/ITx(+) (2.7 ± 0.4 µmol kg(-1) min(-1)) and controls (1.7 ± 0.5 µmol kg(-1) min(-1)), such that it accounted for 70% and 20% of the increased EGP, respectively. Skeletal muscle accounted for similar proportions of the decrease in systemic glucose disposal in controls (49%) and T1DM/ITx(+) (41%). CONCLUSIONS/INTERPRETATION: Partial islet graft function improves hypoglycaemia counterregulation by increasing EGP, largely via lactate GN and decreasing systemic glucose disposal. This may explain the reduction in severe hypoglycaemic events in T1DM/ITx(+) individuals. TRIAL REGISTRATION: ClinicalTrials.gov NCT01668485.

Enhanced oxygen supply improves islet viability in a new bioartificial pancreas.

The current epidemic of diabetes with its overwhelming burden on our healthcare system requires better therapeutic strategies. Here we present a promising novel approach for a curative strategy that may be accessible for all insulin-dependent diabetes patients. We designed a subcutaneous implantable bioartificial pancreas (BAP)-the "ß-Air"-that is able to overcome critical challenges in current clinical islet transplantation protocols: adequate oxygen supply to the graft and protection of donor islets against the host immune system. The system consists of islets of Langerhans immobilized in an alginate hydrogel, a gas chamber, a gas permeable membrane, an external membrane, and a mechanical support. The minimally invasive implantable device, refueled with oxygen via subdermally implanted access ports, completely normalized diabetic indicators of glycemic control (blood glucose intravenous glucose tolerance test and HbA1c) in streptozotocin-induced diabetic rats for periods up to 6 months. The functionality of the device was dependent on oxygen supply to the device as the grafts failed when oxygen supply was ceased. In addition, we showed that the device is immuno-protective as it allowed for survival of not only isografts but also of allografts. Histological examination of the explanted devices demonstrated morphologically and functionally intact islets; the surrounding tissue was without signs of inflammation and showed visual evidence of vasculature at the site of implantation. Further increase in islets loading density will justify the translation of the system to clinical trials, opening up the potential for a novel approach in diabetes therapy.

Improvement of islet function in a bioartificial pancreas by enhanced oxygen supply and growth hormone releasing hormone agonist.

Islet transplantation is a feasible therapeutic alternative for metabolically labile patients with type 1 diabetes. The primary therapeutic target is stable glycemic control and prevention of complications associated with diabetes by reconstitution of endogenous insulin secretion. However, critical shortage of donor organs, gradual loss in graft function over time, and chronic need for immunosuppression limit the indication for islet transplantation to a small group of patients. Here we present a promising approach to address these limitations by utilization of a macrochamber specially engineered for islet transplantation. The s.c. implantable device allows for controlled and adequate oxygen supply and provides immunological protection of donor islets against the host immune system. The minimally invasive implantable chamber normalized blood glucose in streptozotocin-induced diabetic rodents for up to 3 mo. Sufficient graft function depended on oxygen supply. Pretreatment with the growth hormone-releasing hormone (GHRH) agonist, JI-36, significantly enhanced graft function by improving glucose tolerance and increasing ß-cell insulin reserve in rats thereby allowing for a reduction of the islet mass required for metabolic control. As a result of hypervascularization of the tissue surrounding the device, no relevant delay in insulin response to glucose changes has been observed. Consequently, this system opens up a fundamental strategy for therapy of diabetes and may provide a promising avenue for future approaches to xenotransplantation.

Modulation of pancreatic islets-stress axis by hypothalamic releasing hormones and 11beta-hydroxysteroid dehydrogenase.

Corticotropin-releasing hormone (CRH) and growth hormone-releasing hormone (GHRH), primarily characterized as neuroregulators of the hypothalamic-pituitary-adrenal axis, directly influence tissue-specific receptor-systems for CRH and GHRH in the endocrine pancreas. Here, we demonstrate the expression of mRNA for CRH and CRH-receptor type 1 (CRHR1) and of protein for CRHR1 in rat and human pancreatic islets and rat insulinoma cells. Activation of CRHR1 and GHRH-receptor significantly increased cell proliferation and reduced cell apoptosis. CRH stimulated both cellular content and release of insulin in rat islet and insulinoma cells. At the ultrastructural level, CRHR1 stimulation revealed a more active metabolic state with enlarged mitochondria. Moreover, glucocorticoids that promote glucose production are balanced by both 11b-hydroxysteroid dehydrogenase (11ß-HSD) isoforms; 11ß-HSD-type-1 and 11ß-HSD-type-2. We demonstrated expression of mRNA for 11ß-HSD-1 and 11ß-HSD-2 and protein for 11ß-HSD-1 in rat and human pancreatic islets and insulinoma cells. Quantitative real-time PCR revealed that stimulation of CRHR1 and GHRH-receptor affects the metabolism of insulinoma cells by down-regulating 11ß-HSD-1 and up-regulating 11ß-HSD-2. The 11ß-HSD enzyme activity was analyzed by measuring the production of cortisol from cortisone. Similarly, activation of CRHR1 resulted in reduced cortisol levels, indicating either decreased 11ß-HSD-1 enzyme activity or increased 11ß-HSD-2 enzyme activity; thus, activation of CRHR1 alters the glucocorticoid balance toward the inactive form. These data indicate that functional receptor systems for hypothalamic-releasing hormone agonists exist within the endocrine pancreas and influence synthesis of insulin and the pancreatic glucocorticoid shuttle. Agonists of CRHR1 and GHRH-receptor, therefore, may play an important role as novel therapeutic tools in the treatment of diabetes mellitus.

Circulating cytokines are associated with human islet graft function in type 1 diabetes.

Islet cell transplantation has considerable potential as a cure for type 1 diabetes, but recurrent autoimmunity and allograft rejection in which both cytokines play an important role are major obstacles. Using a new approach considering confounders by regression analysis, we investigated circulating cytokines and their association with graft function in type 1 diabetes patients who underwent either simultaneous islet kidney (SIK) or islet after kidney (IAK) transplantation. After transplantation, interleukin (IL)-10 was lower in SIK recipients with subsequent loss of graft function in comparison to recipients maintaining graft function. Before transplantation, high IL-13 and IL-18 concentrations were prospectively associated for subsequent loss of graft function in IAK recipients, whereas in SIK recipients, high macrophage migration inhibitory factor (MIF) concentrations were associated with subsequent loss of graft function. Circulating cytokines are associated with islet graft function in patients with long-standing type 1 diabetes when considering confounders.

Study on pancreatic islet adaptation and gene expression during pregnancy in rats.

During pregnancy, the pancreatic islets undergo major structural and functional changes in response to increased peripheral resistance to insulin. In this study, we investigated the adaptive changes of the pancreatic islet beta-cell mass during pregnancy in rats, and explored profiles of islet gene expression at various stages of pregnancy. Some differentially expressed genes were verified by RT-PCR and Real-time PCR. Our results showed that compared with the non-pregnant control group, insulin synthesis, glucose-stimulated insulin secretion, islet beta-cell proliferation, and islet size were all increased in pregnant rats. The study also demonstrated that expression of several-hundred islet genes were changed during pregnancy, especially at day 14.5. The differentially expressed genes identified were distributed into eight main categories according to their biological functions: (1) genes involved in apoptosis or tumor; (2) genes related to binding; (3) genes involved in metabolism; (4) genes related to cell cycle; (5) genes for signal transducer activity; (6) genes related to structural molecule activity; (7) genes involved in transcription regulator activity; (8) genes for transporter activity. Among these genes, regenerating islet-derived 3 alpha (Reg3a) was remarkably increased during pregnancy. We hypothesize that differentially expressed genes may play an important role in adaptation of pancreatic islets during pregnancy in rats. In addition, the markedly increased expression of gene Reg3a is probably related to islet regeneration.

Agonist of growth hormone-releasing hormone as a potential effector for survival and proliferation of pancreatic islets.

Therapeutic strategies for transplantation of pancreatic islet cells are urgently needed to expand beta-cell mass by stimulating islet cell proliferation and/or prolonging islet cell survival. Control of the islets by different growth factors provides a potential venue for augmenting beta-cell mass. In the present study, we show the expression of the biologically active splice variant-1 (SV-1) of growth hormone-releasing hormone (GHRH) receptor in rat insulinoma (INS-1) cells as well as in rat and human pancreatic islets. In studies in vitro of INS-1 cells, the GHRH agonist JI-36 caused a significant increase in cell proliferation and a reduction of cell apoptosis. JI-36 increased islet size and glucose-stimulated insulin secretion in isolated rat islets after 48-72 h. At the ultrastructural level, INS-1 cells treated with agonist JI-36 revealed a metabolic active stimulation state with increased cytoplasm. Coincubation with the GHRH antagonist MIA-602 reversed the actions of the agonist JI-36, indicating the specificity of this agonist. In vivo, the function of pancreatic islets was assessed by transplantation of rat islets under the kidney capsule of streptozotocin-induced diabetic non-obese diabetic-severe combined immunodeficiency (NOD-SCID) mice. Islets treated with GHRH agonist JI-36 were able to achieve normoglycemia earlier and more consistently than untreated islets. Furthermore, in contrast to diabetic animals transplanted with untreated islets, insulin response to an i.p. glucose tolerance test (IPGTT) in animals receiving islets treated with agonist Jl-36 was comparable to that of normal healthy mice. In conclusion, our study provides evidence that agonists of GHRH represent a promising pharmacological therapy aimed at promoting islet graft growth and proliferation in diabetic patients.

Transmission infrared spectroscopy of whole blood--complications for quantitative analysis from leucocyte adhesion during continuous monitoring.

Infrared spectroscopy has been applied to analyse glucose and cellular components in whole blood with the aim of developing an online clinical diagnostic and monitoring modality. Leucocyte adsorption onto the CaF(2) windows was observed over a period of several hours under continuous blood flow using a transmission cell of 30 mum path length. This build-up of cellular material on the windows is responsible for diminishing the sample path length under the flow conditions chosen. The adsorption dynamics have been characterised and their impact on glucose monitoring is reported. For short-term monitoring (<2 hours) a standard error of prediction of 11 mg/dL with human citrated blood samples from three different subjects was achieved. Furthermore, the leucocyte build-up was also reported for porcine EDTA blood monitoring. Consequences and testing opportunities with regard to the first stages in the immune cell reaction to the exposure of body-foreign materials to anticoagulated whole blood are discussed.

Is pancreatic diabetes (type 3c diabetes) underdiagnosed and misdiagnosed?

Exocrine pancreatic insufficiency is frequently associated with diabetes, with high prevalence in both insulin-dependent or insulin-independent patients. Exocrine pancreatic failure has often been perceived as a complication of diabetes. In contrast, recent clinical observations lead to the notion that nonendocrine pancreatic disease is a critical factor for development rather than a sequel to diabetes. The incidence of diabetes caused by exocrine pancreatic disease appears to be underestimated and may comprise 8% or more of the general diabetic patient population. Nonendocrine pancreas disease can cause diabetes by multiple mechanisms. Genetic defects have been characterized, resulting in a syndrome of both exocrine and endocrine failure. Regulation of beta-cell mass and physiological incretin secretion are directly dependent on normal exocrine function. Algorithms for diagnosis and therapy of diabetes should therefore address both endocrine and exocrine pancreatic function.

Islet cell transplantation today.

INTRODUCTION: Long-term studies strongly suggest that tight control of blood glucose can prevent the development and retard the progression of chronic complications of type 1 diabetes mellitus. In contrast to conventional insulin treatment, replacement of a patient's islets of Langerhans either by pancreas organ transplantation or by isolated islet transplantation is the only treatment to achieve a constant normoglycemic state and avoiding hypoglycemic episodes, a typical adverse event of multiple daily insulin injections. However, the cost of this benefit is still the need for immunosuppressive treatment of the recipient with all its potential risks. MATERIALS AND METHODS: Islet cell transplantation offers the advantage of being performed as a minimally invasive procedure in which islets can be perfused percutaneously into the liver via the portal vein. Between January 1990 and December 2004, 458 pancreatic islet transplants worldwide have been reported to the International Islet Transplant Registry (ITR) at our Third Medical Department, University of Giessen/Germany. RESULTS: Data analysis of islet cell transplants performed in the last 5 years (1999-2004) shows at 1 year after adult islet transplantation a patient survival rate of 97%, a functioning islet graft in 82% of the cases, whereas insulin independence was meanwhile achieved in 43% of the cases. However, using a novel protocol established by the Edmonton Center/Canada, the insulin independence rates have improved significantly reaching meanwhile a 50-80% level. CONCLUSION: Finally, the concept of islet cell or stem cell transplantation is most attractive, as it offers many perspectives: islet cell availability could become unlimited and islet or stem cells my be transplanted without life-long immunosuppressive treatment of the recipient, just to mention two of them.

Characterization of somatostatin receptor subtype-specific regulation of insulin and glucagon secretion: an in vitro study on isolated human pancreatic islets.

INTRODUCTION: Pancreatic A- and B-cells express somatostatin receptors (SSTRs). Five pharmacologically distinct SSTR subtypes are known (SSTR1-SSTR5). In rodents, SSTR2 inhibits glucagon secretion, whereas SSTR5 suppresses the release of insulin. Human pancreatic A- and B-cells express SSTR1-3 and SSTR5; however, their contribution to the regulation of glucagon and insulin secretion is not well known. AIM OF THE STUDY: The goal of this study was to characterize the role of individual SSTR subtypes in regulating human glucagon and insulin secretion in vitro. METHODS: Human pancreatic islets were isolated from healthy donors and incubated with somatostatin, SSTR1-3-selective and SSTR5-selective agonists, or an SSTR2-selective antagonist (DC-41-33). Stimulation of insulin secretion was induced by glucose (10, 20 mm) alone or in combination with 10 nm exendin-4 or 10 mm L-arginine. Glucagon secretion was induced by 20 mm L-arginine. Basal secretion of insulin and glucagon was measured at 2.8 or 3.3 mm glucose. RESULTS: SSTR1-, SSTR2-, and SSTR5-selective agonists inhibited insulin secretion with the following order of potency: SSTR2 (EC50, 0.08 nm) > SSTR5 (EC50, 5.3 nm) > SSTR1 (EC50, 35 nm). Glucagon secretion was inhibited by SSTR-selective agonists with the following order of potency: SSTR2 (EC50, 0.05 nm) > SSTR1 (EC50, 1.8 nm) > SSTR5 (EC50, 28 nm). DC-41-33 dose-dependently reversed the effects of the SSTR2-selective agonist on insulin and glucagon secretion. CONCLUSION: Our study demonstrates that SSTR2-agonist is the most potent inhibitor of insulin and glucagon secretion from isolated human pancreatic islets. Furthermore, we identify SSTR1- and SSTR5-selective agonists as additional inhibitors of insulin and glucagon secretion from human pancreas.

International trial of the Edmonton protocol for islet transplantation.

BACKGROUND: Islet transplantation offers the potential to improve glycemic control in a subgroup of patients with type 1 diabetes mellitus who are disabled by refractory hypoglycemia. We conducted an international, multicenter trial to explore the feasibility and reproducibility of islet transplantation with the use of a single common protocol (the Edmonton protocol). METHODS: We enrolled 36 subjects with type 1 diabetes mellitus, who underwent islet transplantation at nine international sites. Islets were prepared from pancreases of deceased donors and were transplanted within 2 hours after purification, without culture. The primary end point was defined as insulin independence with adequate glycemic control 1 year after the final transplantation. RESULTS: Of the 36 subjects, 16 (44%) met the primary end point, 10 (28%) had partial function, and 10 (28%) had complete graft loss 1 year after the final transplantation. A total of 21 subjects (58%) attained insulin independence with good glycemic control at any point throughout the trial. Of these subjects, 16 (76%) required insulin again at 2 years; 5 of the 16 subjects who reached the primary end point (31%) remained insulin-independent at 2 years. CONCLUSIONS: Islet transplantation with the use of the Edmonton protocol can successfully restore long-term endogenous insulin production and glycemic stability in subjects with type 1 diabetes mellitus and unstable control, but insulin independence is usually not sustainable. Persistent islet function even without insulin independence provides both protection from severe hypoglycemia and improved levels of glycated hemoglobin. (ClinicalTrials.gov number, NCT00014911 [ClinicalTrials.gov].).

Glucose-dependent expansion of pancreatic beta-cells by the protein p8 in vitro and in vivo.

p8 protein expression is known to be upregulated in the exocrine pancreas during acute pancreatitis. Own previous work revealed glucose-dependent p8 expression also in endocrine pancreatic beta-cells. Here we demonstrate that glucose-induced INS-1 beta-cell expansion is preceded by p8 protein expression. Moreover, isopropylthiogalactoside (IPTG)-induced p8 overexpression in INS-1 beta-cells (p8-INS-1) enhances cell proliferation and expansion in the presence of glucose only. Although beta-cell-related gene expression (PDX-1, proinsulin I, GLUT2, glucokinase, amylin) and function (insulin content and secretion) are slightly reduced during p8 overexpression, removal of IPTG reverses beta-cell function within 24 h to normal levels. In addition, insulin secretion of p8-INS-1 beta-cells in response to 0-25 mM glucose is not altered by preceding p8-induced beta-cell expansion. Adenovirally transduced p8 overexpression in primary human pancreatic islets increases proliferation, expansion, and cumulative insulin secretion in vitro. Transplantation of mock-transduced control islets under the kidney capsule of immunosuppressed streptozotocin-diabetic mice reduces blood glucose and increases human C-peptide serum concentrations to stable levels after 3 days. In contrast, transplantation of equal numbers of p8-transduced islets results in a continuous decrease of blood glucose and increase of human C-peptide beyond 3 days, indicating p8-induced expansion of transplanted human beta-cells in vivo. This is underlined by a doubling of insulin content in kidneys containing p8-transduced islet grafts explanted on day 9. These results establish p8 as a novel molecular mediator of glucose-induced pancreatic beta-cell expansion in vitro and in vivo and support the notion of existing beta-cell replication in the adult organism.

Vascular endothelial growth factor increases functional beta-cell mass by improvement of angiogenesis of isolated human and murine pancreatic islets.

BACKGROUND: Blood flow is impaired in islet transplants, but there is conflicting evidence on improving the outcome by promoting vascularization. We previously reported that islet endothelial cells (EC) possess significant angiogenic capacity. METHODS: To further address this issue, we studied human islets in culture under hypoxic conditions. Moreover, we used a transgene mouse model with human vascular endothelial growth factor (VEGF) production in beta-cells under the control of the rat insulin promoter (RIP) to stimulate islet EC proliferation. RESULTS: Subsequent to a hypoxic stimulus, islets responded with specific expression patterns of VEGF and fibroblast growth factor; however, this was not sufficient to prevent the decay of islet EC. VEGF release of RIP-VEGF transgenic islets was controlled by glucose and resulted in the formation of sprouts. When transplanted to the kidney capsule of diabetic mice, RIP-VEGF islets significantly enhanced microvascular density and functional blood flow to the graft compared with controls. CONCLUSIONS: Optimized angiogenesis of islet transplants resulted in greater availability of insulin caused by beta-cell proliferation and a significantly higher percentage (90% versus 20%) of mice cured from diabetes.

Influence of collagenase loading on long-term preservation of pig pancreas by the two-layer method for subsequent islet isolation.

BACKGROUND: The introduction of the two-layer method (TLM) for long-term human pancreas preservation revealed the enormous potential of TLM to improve graft function of isolated islets. It is still unclear whether pig islets can be successfully isolated from pancreases after prolonged cold ischemia. To clarify this question, pig pancreases were subjected to 7-hour preservation by University of Wisconsin solution (UWS) storage or TLM. Another aim was to verify whether TLM can be synergistically combined with intraductal collagenase injection before cold storage. METHODS: After intraductal flush with UWS, organs were distended with 4.4 PZ-U/g of UWS-dissolved collagenase NB-8 and neutral protease adjusted to respectively 1.1, 0.2, 0.5, or 0.8 DMC-U/g for pancreases freshly procured (n=6) or distended with enzymes before (TLM preloaded, n=7) or after cold storage (UWS storage, n=4; TLM postloaded, n=10). RESULTS: Purified islet yield decreased from 429,200+/-86,700 islet equivalents (IEQ) in unstored pancreases to respectively 37,670+/-19620, 210,400+/-22900 and 238,000+/-26600 IEQ in UWS-stored (P<0.01), TLM-preloaded, or postloaded organs (P<0.05). Purity (>90%), viability (>95%), and insulin content were not different between groups. Islets from UWS-stored pancreases fragmented extensively, preventing further assessment of in vivo function. Compared with other experimental groups, islets from TLM-preloaded organs were characterized by enhanced basal and stimulated insulin release. Sustained normoglycemia was observed in diabetic nude mice transplanted with islets from TLM-postloaded or unstored pancreases in contrast with transient function in TLM-preloaded islets. CONCLUSIONS: This study demonstrates that significant amounts of intact pig islets can be isolated after prolonged pancreas preservation by TLM. Enzyme administration before TLM preservation decreases islet graft function.

Successful pancreas preservation by a perfluorocarbon-based one-layer method for subsequent pig islet isolation.

BACKGROUND: The oxygenation of human pancreas by the two-layer method (TLM) during cold storage was recently established for clinical islet transplantation. Simplification of TLM would facilitate the application of perfluorocarbon (PFC) as a regularly used preservation solution for subsequent islet transplantation. The present study examined whether PFC can be used in a one-layer method (OLM) for long-term pancreas preservation before isolation of adult pig islets. METHODS: Resected pancreases were intraductally flushed with cold University of Wisconsin solution and immediately processed (n=6) or subjected to 7-hour storage by OLM (n=8) or TLM (n=10). Subsequently, pancreases were intraductally distended with collagenase NB-8 supplemented with neutral protease. Isolation and purification were performed as previously described. RESULTS: Compared with unstored pancreases (3,670+/-740 islet equivalents [IEQ]) purified islet yield in TLM-stored organs (2,080+/-290 IEQ, P<0.05) was significantly decreased in contrast with OLM-preserved pancreases (3,110+/-520 IEQ, NS). No differences were observed between groups regarding purity (>90%), trypan-blue exclusion (>95%), adenosine triphosphate content, and mitochondrial viability of islets. Stimulation index during static glucose incubation (20 vs. 2.8 mm) was decreased after storage by TLM (1.81+/-0.20, P<0.05) but not by OLM (2.27+/-0.57) if compared with unstored pancreases (2.47+/-0.36). However, transplantation into diabetic nude mice resulted in sustained normoglycemia of recipients of either group until nephrectomy of graft-bearing kidneys was performed. CONCLUSIONS: This study demonstrates that PFC alone can be used in a one-layer procedure for successful pig-pancreas preservation. This simplification can facilitate the broad application of PFC as pancreas preservation solution without reducing its benefits demonstrated by TLM.

[Apoptosis induced by free fatty acids]. / Fettsäureinduzierte beta-Zell-Apoptose.

BACKGROUND AND AIMS: Elevated free fatty acid (FFA) levels are believed to be one of the major contributing factors in the pathogenesis of type 2 diabetes. FFAs enhance peripheral insulin resistance, promote beta-cell dysfunction, and trigger beta-cell death. The purpose of this study was to investigate whether the lipoapoptotic effect of FFAs is determined by the degree of saturation and the chain length, and which pathways might be involved. MATERIAL AND METHODS: After incubation of RIN1046-38 rat insulinoma cells and primary human islet cells with different FFAs, apoptosis was assessed by flow cytometry (sub-G1-DNA formation) and TUNEL assay, respectively. Intracellular distribution of protein kinase C-delta (PKC-delta) was determined by confocal laser microscopy. RESULTS: In RIN1046-38 cells, the sub-G1 fraction increased 40-fold over basal (0.5% to approximately 20%) after treatment with the saturated FFAs palmitate and stearate (1 mM, 24 h), while the monounsaturated FFAs palmitoleate and oleate as well as the polyunsaturated FFA linoleate had no proapoptotic effect. Unsaturated FFAs even more prevented palmitate- and stearate-induced apoptosis completely. Furthermore, these results could be confirmed in primary beta-cells of human islets. Investigating potential underlying signaling pathways, it was found that only the saturated FFAs were able to induce nuclear translocation of PKC-delta. CONCLUSIONS: FFAs exert different effects on beta-cell survival. Saturated FFAs turned out to be pro-apoptotic, whereas unsaturated FFAs protected from saturated FFA-induced apoptosis. Furthermore, our data suggest an involvement of PKC-delta in apoptosis induced by saturated FFAs.

Protein kinase C delta activation and translocation to the nucleus are required for fatty acid-induced apoptosis of insulin-secreting cells.

Insulin resistance as well as pancreatic beta-cell failure can be induced by elevated free fatty acid (FFA) levels. We studied the mechanisms of FFA-induced apoptosis in rat and human beta-cells. Chronic treatment with high physiological levels of saturated fatty acids (palmitate and stearate), but not with monounsaturated (palmitoleate and oleate) or polyunsaturated fatty acids (linoleate), triggers apoptosis in approximately 20% of cultured RIN1046-38 cells. Apoptosis restricted to saturated FFAs was also observed in primary cultured human beta-cells, suggesting that this mechanism is potentially relevant in vivo in humans. To further analyze FFA-induced signaling pathways leading to apoptosis, we used RIN1046-38 cells. Apoptosis was accompanied by a rapid (within 15 min) nuclear translocation of protein kinase C (PKC)-delta and subsequent lamin B1 disassembly. This translocation was impaired by the phospholipase C inhibitor U-73122, which also substantially reduced apoptosis. Furthermore, lamin B1 disassembly and apoptosis were decreased by cell transfection with a dominant-negative mutant form of PKC-delta. These data suggest that nuclear translocation and kinase activity of PKC-delta are both necessary for saturated fatty acid-induced apoptosis.

Hyperthermic preconditioning protects pig islet grafts from early inflammation but enhances rejection in immunocompetent mice.

The induction of heat shock proteins (HSP) protects isolated islet cells against the cytotoxicity of inflammatory mediators in vitro. Very little information is available about the effect of HSP overexpression on function of preconditioned islet grafts. The present study investigated the function of heat-exposed pig islets after transplantation into immunocompetent mice in comparison with in vitro resistance against inflammatory mediators. Pig islets were preconditioned at 43 degrees C or sham treated prior to subcapsular transplantation into diabetic C57/Bl6j mice. Nondiabetic mice simultaneously receiving preconditioned and control islets were subjected to bilateral nephrectomy for determination of pig insulin. Resistance against H2O2, NO, human Il-1beta, IFN-gamma, or TNF-alpha was assessed by trypan blue exclusion and insulin determination. Heat-induced protein expression was confirmed by Western blot analysis. Graft preconditioning increased resistance against H2O2, NO, or cytokines (p < 0.05) but decreased survival in nondiabetic mice (p < 0.05) and function in diabetic mice (p < 0.01). Upregulation of caspase-3 activity as well as Bax, Fas, FasL, and DFF expression (p < 0.05) indicated simultaneous induction of apoptosis. The coexpression of HSP and proapoptotic proteins reveals the dual character of the stress response simultaneously starting mechanisms for protection and apoptosis. In vitro assays seem to reflect only insufficiently the situation of islets after transplantation.

Different role of saturated and unsaturated fatty acids in beta-cell apoptosis.

It is believed that free fatty acids contribute to the pathogenesis of type 2 diabetes in humans. We have recently shown that lipoapoptosis of human beta-cells is specifically induced by saturated fatty acids while unsaturated had no effect. In the present study we tested the effect of co-incubation of different saturated and unsaturated free fatty acids on lipoapoptosis in beta-cells. RIN1046-38 cells and isolated human beta-cells were incubated with combinations of saturated fatty acids (palmitate, stearate) and mono- or polyunsaturated fatty acids (palmitoleate, oleate, and linoleate). Cells were incubated for 24-72 h with 1mM fatty acids. All unsaturated fatty acids tested completely prevented palmitate- or stearate-induced apoptosis of rat and human beta-cells as assessed by flow cytometric cell cycle analysis and TUNEL assay. This might suggest that apoptosis in vivo is predominantly determined by the content of unsaturated fatty acids in a mixed fatty acid pool.