Heme oxygenase-1 fused to a TAT peptide transduces and protects pancreatic beta-cells

Transplantation of islets is becoming an established method for treating type 1 diabetes. However, viability of islets is greatly affected by necrosis/apoptosis induced by oxidative stress and other insults during isolation and subsequent in vitro culture. Expression of cytoprotective proteins, such as heme oxygenase-1 (HO-1), reduces the deleterious effects of oxidative stress in transplantable islets. We have generated a fusion protein composed of HO-1 and TAT protein transduction domain (TAT/PTD), an 11-aa cell penetrating peptide from the human immunodeficiency virus TAT protein. Transduction of TAT/PTD-HO-1 to insulin-producing cells protects against TNF-alpha-mediated cytotoxicity. TAT/PTD-HO-1 transduction to islets does not impair islet physiology, as assessed by reversion of chemically induced diabetes in immunodeficient mice. Finally, we report that transduction of HO-1 fusion protein into islets improves islet viability in culture. This approach might have a positive impact on the availability of islets for transplantation.     

Characterization of the mouse pancreatic islet proteome and comparative analysis with other mouse tissues

The pancreatic islets of Langerhans, and especially the insulin-producing beta cells, play a central role in the maintenance of glucose homeostasis. Alterations in the expression of multiple proteins in the islets that contribute to the maintenance of islet function are likely to underlie the pathogenesis of types 1 and 2 diabetes. To identify proteins that constitute the islet proteome, we provide the first comprehensive proteomic characterization of pancreatic islets for mouse, the most commonly used animal model in diabetes research. Using strong cation exchange fractionation coupled with reversed phase LC-MS/MS we report the confident identification of 17,350 different tryptic peptides covering 2612 proteins having at least two unique peptides per protein. The data set also identified approximately 60 post-translationally modified peptides including oxidative modifications and phosphorylation. While many of the identified phosphorylation sites corroborate those previously known, the oxidative modifications observed on cysteinyl residues reveal potentially novel information suggesting a role for oxidative stress in islet function. Comparative analysis with 15 available proteomic data sets from other mouse tissues and cells revealed a set of 133 proteins predominantly expressed in pancreatic islets. This unique set of proteins, in addition to those with known functions such as peptide hormones secreted from the islets, contains several proteins with as yet unknown functions. The mouse islet protein and peptide database accessible at (http://ncrr.pnl.gov), provides an important reference resource for the research community to facilitate research in the diabetes and metabolism fields.     

Effect of the pancreatic digestion with liberase versus collagenase on the yield, function and viability of neonatal rat pancreatic islets

Many obstacles hinder the clinical application of pancreatic islet transplantation as a cure for diabetes mellitus. One of them is the suitable isolation method of sufficient number of healthy islets for transplantation. In this context, liberase enzyme was developed as a purified form of the traditional collagenase. It was the aim of this study to investigate the effect of liberase-digestion on the yield, function and viability of neonatal rat islets, and to compare the new enzyme with the collagenase. Glucose-stimulated insulin secretion was measured as indication of the function, insulin content as indication for the synthetic activity of islet cells and DNA as an indication of cell viability. The results showed no difference between islets isolated either with collagenase or liberase. Glucose stimulated similarly the insulin secretion in both. Stimulation index tended, without significance, to be higher (55%) in liberase-isolated islets compared with the collagenase islets (49%). The viability of both was similar. The insulin synthesis (content) tended also to be better in liberase-isolated islets. It could be concluded that liberase could be non-significantly preferred in the isolation of neonatal rat islets in comparison with collagenase.     

Glutathione Ethyl Ester Supplementation during Pancreatic Islet Isolation Improves Viability and Transplant Outcomes in a Murine Marginal Islet Mass Model

Background

The success of pancreatic islet transplantation still faces many challenges, mainly related to cell damage during islet isolation and early post-transplant. The increased generation of reactive oxygen species (ROS) during islet isolation and the consumption of antioxidant defenses appear to be an important pathway related to islet damage.

Methodology/Principal Findings

In the present study we evaluated whether supplementation of glutathione-ethyl-ester (GEE) during islet isolation could improve islet viability and transplant outcomes in a murine marginal islet mass model. We also cultured human islets for 24 hours in standard CMRL media with or without GEE supplementation. Supplementation of GEE decreased the content of ROS in isolated islets, leading to a decrease in apoptosis and maintenance of islet viability. A higher percentage of mice transplanted with a marginal mass of GEE treated islets became euglycemic after transplant. The supplementation of 20 mM GEE in cultured human islets significantly reduced the apoptosis rate in comparison to untreated islets.

Conclusions/Significance

GEE supplementation was able to decrease the apoptosis rate and intracellular content of ROS in isolated islets and might be considered a potential intervention to improve islet viability during the isolation process and maintenance in culture before islet transplantation.     

Long-term normoglycemia in pancreatectomized dogs transplanted with frozen/thawed pancreatic islets

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

Optimizing Conditions for Rat Pancreatic Islets Isolation

Many procedures have been described for rat pancreatic islet isolation. Several factors contribute to the pancreatic islet isolation outcome. One of the main problems in islet isolation procedure is the formation of a viscouse, gellike structure during collagenase digestion which entraps the free islets and decrease islet yield after density gradient purification. This issue has not been addressed in most techniques described for rat islet isolation. We examined effect of various factors to eliminate formation of gellike material and improve the islets yields. Islet isolation was performed on 26 adult male Wistar Albino rats weighing between 280 and 350 g. We have observed that several factors affect pancreatic islet isolation. Optimum Collagenase enzyme concentration, maintaining pH range between 7.7 and 7.9 in digestion solution, incubation temperature at 38±1 °C and addition of Calcium ion decreased the formation of gellike materials and increased islet yield. Addition of Glycerol as a gelatin solvent has also been helpful in the reduction or complete elimination of gellike material. Precise optimization of rat islet isolation procedure is useful to improve the islet yield in islet transplantation studies.     

A Practical Guide to Rodent Islet Isolation and Assessment

Pancreatic islets of Langerhans secrete hormones that are vital to the regulation of blood glucose and are, therefore, a key focus of diabetes research. Purifying viable and functional islets from the pancreas for study is an intricate process. This review highlights the key elements involved with mouse and rat islet isolation, including choices of collagenase, the collagenase digestion process, purification of islets using a density gradient, and islet culture conditions. In addition, this paper reviews commonly used techniques for assessing islet viability and function, including visual assessment, fluorescent markers of cell death, glucose-stimulated insulin secretion, and intracellular calcium measurements. A detailed protocol is also included that describes a common method for rodent islet isolation that our laboratory uses to obtain viable and functional mouse islets for in vitro study of islet function, beta-cell physiology, and in vivo rodent islet transplantation. The purpose of this review is to serve as a resource and foundation for successfully procuring and purifying high-quality islets for research purposes.     

Application of magnetic particle tracking velocimetry to quadrupole magnetic sorting of porcine pancreatic islets

Magnetic isolation is a promising method for separating and concentrating pancreatic islets of Langerhans for transplantation in Type 1 diabetes patients. We are developing a continuous magnetic islet sorter to overcome the restrictions of current purification methods that result in limited yield and viability. In Quadrupole Magnetic Sorting (QMS) islets are magnetized by infusing superparamagnetic microbeads into islets' vasculature via arteries that serve the pancreas. The performance of the islet sorter depends on the resulting speed of the islets in an applied magnetic field, a property known as magnetophoretic mobility. Essential to the design and successful operation of the QMS is a method to measure the magnetophoretic mobilities of magnetically infused islets. We have adapted a Magnetic Particle Tracking Velocimeter (MPTV) to measure the magnetophoretic mobility of particles up to 1,000 μm in diameter. Velocity measurements are performed in a well-characterized uniform magnetic energy gradient using video imaging followed by analysis of the video images with a computer algorithm that produces a histogram of absolute mobilities. MPTV was validated using magnetic agarose beads serving as islet surrogates and subjecting them to QMS. Mobility distributions of labeled porcine islets indicated that magnetized islets have sufficient mobility to be captured by the proposed sorting method, with this result confirmed in test isolations of magnetized islets.     

Fibroblast populated collagen matrix promotes islet survival and reduces the number of islets required for diabetes reversal

Islet transplantation represents a viable treatment for type 1 diabetes. However, due to loss of substantial mass of islets early after transplantation, islets from two or more donors are required to achieve insulin independence. Islet-extracellular matrix disengagement, which occurs during islet isolation process, leads to subsequent islet cell apoptosis and is an important contributing factor to early islet loss. In this study, we developed a fibroblast populated collagen matrix (FPCM) as a novel scaffold to improve islet cell viability and function post-transplantation. FPCM was developed by embedding fibroblasts within type-I collagen and used as scaffold for islet grafts. Viability and insulin secretory function of islets embedded within FPCM was evaluated in vitro and in a syngeneic murine islet transplantation model. Islets embedded within acellular matrix or naked islets were used as control. Islet cell survival and function was markedly improved particularly after embedding within FPCM. The composite scaffold significantly promoted islet isograft survival and reduced the critical islet mass required for diabetes reversal by half (from 200 to 100 islets per recipient). Fibroblast embedded within FPCM produced fibronectin and growth factors and induced islet cell proliferation. No evidence of fibroblast over-growth within composite grafts was noticed. These results confirm that FPCM significantly promotes islet viability and functionality, enhances engraftment of islet grafts and decreases the critical islet mass needed to reverse hyperglycemia. This promising finding offers a new approach to reducing the number of islet donors per recipient and improving islet transplant outcome.     

Method for isolation of pancreatic blood vessels,their culture and coculture with islets of langerhans

The only cure available for Type 1 diabetes involves the transplantation of islets of Langerhans isolated from donor organs. However, success rates are relatively low. Disconnection from vasculature upon isolation and insufficient rate of revascularization upon transplantation are thought to be a major cause, as islet survival and function depend on extensive vascularization. Research has thus turned toward the development of pretransplantation culture techniques to enhance revascularization of islets, so far with limited success. With the aim to develop a technique to enhance islet revascularization, this work proposes a method to isolate and culture pancreas-derived blood vessels. Using a mild multistep digestion method, pancreatic blood vessels were retrieved from whole murine pancreata and cultured in collagen Type 1. After 8 days, 50% of tissue explants had formed anastomosed microvessels which extended up to 300 μm from the explant tissue and expressed endothelial cell marker CD31 but not ductal marker CK19. Cocultures with islets of Langerhans revealed survival of both tissues and insulin expression by islets up to 8 days post-embedding. Microvessels were frequently found to encapsulate islets, however no islet penetration could be detected. This study reports for the first time the isolation and culture of pancreatic blood vessels. The methods and results presented in this work provide a novel explant culture model for angiogenesis and tissue engineering research with relevance to islet biology. It opens the door for in vivo validation of the potential of these pancreatic blood vessel explants to improve islet transplantation therapies. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2745, 2019.     

In vivo monitoring of pancreatic beta-cells in a transgenic mouse model

We generated a transgenic mouse model (RIP-luc) for the in vivo monitoring of pancreatic islet mass and function in response to metabolic disease. Using the rat insulin promoter fused to firefly luciferase, and noninvasive technology to detect luciferase activity, we tracked changes in reporter signal during metabolic disease states and correlated the changes in luciferase signal with metabolic status of the mouse. Transgene expression was found to be specific to the pancreatic islets in this transgenic model. Basal transgene expression was tracked in male and female mice fed either a chow or a high-fat diet and in response to treatment with streptozotocin. Pancreatic bioluminescent signal increased in mice fed a high-fat diet compared with chow-fed animals. In a model of chemically induced diabetes, the bioluminescent signal decreased in accordance with the onset of diabetes and reduction of islet beta-cell number. Preliminary studies using islets transplanted from this transgenic model suggest that in vivo image analysis can also be used to monitor transplanted islet viability and survival in the host. This transgenic model is a useful tool for in vivo studies of pancreatic beta-cells and as a donor for islet transplantation studies.     

Hexose metabolism in pancreatic islets. Participation of Ca2(+)-sensitive 2-ketoglutarate dehydrogenase in the regulation of mitochondrial function

A rise in extracellular D-glucose concentration results in a preferential and Ca2(+)-dependent stimulation of mitochondrial oxidative events in pancreatic islet cells. The possible participation of Ca2(+)-dependent mitochondrial dehydrogenases, especially 2-ketoglutarate dehydrogenase, in such an unusual metabolic situation was explored in intact islets, islet homogenates and isolated islet mitochondria. In intact islets exposed to a high concentration of D-glucose, the removal of extracellular Ca2+ impaired D-[6-14C]glucose oxidation whilst failing to affect the cytosolic or mitochondrial ATP/ADP ratios. In islet homogenates, the activity of 2-ketoglutarate dehydrogenase displayed exquisite Ca2(+)-dependency, the presence of Ca2+ causing a 10-fold increase in affinity for 2-ketoglutarate. In intact islet mitochondria, the oxidation of 2-[1-14C]ketoglutarate also increased as a function of extramitochondrial Ca2+ availability. Moreover, prior stimulation of intact islets by D-glucose resulted in an increased capacity of mitochondria to oxidize 2-[1-14C]ketoglutarate. The absence of extracellular Ca2+ during the initial stimulation of intact islets impaired but did not entirely suppress such a memory phenomenon. It is proposed that the mitochondrial accumulation of Ca2+ in nutrient-stimulated islets indeed accounts, in part at least, for the preferential stimulation of mitochondrial oxidative events in this fuel-sensor organ.     

Effects of Excess Dietary Fluoride on Serum Biochemical Indices,Egg Quality,and Concentrations of Fluoride in Soft Organs,Eggs, and Serum of Laying Hens

According to undiscovered toxicity and safety of magnesium oxide nanoparticles (MgO NPs) in isolated pancreatic islet cells, this study was designed to examine the effects of its various concentrations on a time-course basis on the oxidative stress, viability, and function of isolated islets of rat’s pancreas. Pancreatic islets were isolated and exposed to different MgO NP (<100 nm) concentrations within three different time points. After that, oxidative stress biomarkers were investigated and the best exposure time was selected. Then, safety of MgO NPs was investigated by flow cytometry and fluorescent staining, and levels of insulin secretion and caspase activity were measured. The results illustrated a considerable decrease in oxidative stress markers such as reactive oxygen species (ROS) and lipid peroxidation (LPO) levels of pancreatic islets which were treated by MgO NPs for 24 h. Also, in that time of exposure, cell apoptosis investigation by flow cytometry and insulin test showed that MgO NPs, in a concentration of 100 μg/ml, decreased the rate of apoptotic cells via inhibiting caspase-9 activity and made a significant increase in the level of insulin secretion. Data of function and apoptosis biomarkers correlated with each other. It is concluded that the use of MgO NPs in concentration of as low as 100 μg/ml can induce antiapoptotic, antioxidative, and antidiabetic effects in rat pancreatic islets, which support its possible benefit in islet transplantation procedures.     

Immuno-Isolation of Pancreatic Islet Allografts Using Pegylated Nanotherapy Leads to Long-Term Normoglycemia in Full MHC Mismatch Recipient Mice

Two major hurdles need to be surmounted for cell therapy for diabetes: (i) allo-immune rejection of grafted pancreatic islets, or stem/precursor cell-derived insulin-secreting cells; and (ii) continuing auto-immunity against the diabetogenic endogenous target antigen. Nanotherapeutics offer a novel approach to overcome these problems and here we ask if creation of “stealth” islets encapsulated within a thin cage of pegylated material of 100–200 nanometers thick provides a viable option for islet transplantation. The aims of this study were to test islet viability and functionality following encapsulation within the pegylated cage, and functional efficacy in vivo in terms of graft-derived control of normoglycemia in diabetic mice. We first demonstrated that pegylation of the islet surface, plus or minus nanoparticles, improved long-term islet viability in vitro compared to non-pegylated (naked) control islets. Moreover, pegylation of the islets with nanoparticles was compatible with glucose-stimulated insulin secretion and insulin biogenesis. We next looked for functionality of the created “stealth” DBA/2 (H-2^d) islets in vivo by comparing glycemic profiles across 4 groups of streptozotozin-induced diabetic C57BL/6 (H-2^b) recipients of (i) naked islets; (ii) pegylated islets; (iii) pegylated islets with nanoparticles (empty); and (iv) pegylated islets with nanoparticles loaded with a cargo of leukemia inhibitory factor (LIF), a factor both promotes adaptive immune tolerance and regulates pancreatic β cell mass. Without any other treatment, normoglycemia was lost after 17 d (+/−7.5 d) in control group. In striking contrast, recipients in groups (ii), (iii), and (iv) showed long-term (>100 d) normoglycemia involving 30%; 43%, and 57% of the recipients in each respective group. In conclusion, construction of “stealth” islets by pegylation-based nanotherapeutics not only supports islet structure and function, but also effectively isolates the islets from immune-mediated destruction. The added value of nanoparticles to deliver immune modulators plus growth factors such as LIF expands the potential of this novel therapeutic approach to cell therapy for diabetes.     

Protection of rat pancreatic islet function and viability by coculture with rat bone marrow-derived mesenchymal stem cells

The maintenance of viable and functional islets is critical in successful pancreatic islet transplantation from cadaveric sources. During the isolation procedure, islets are exposed to a number of insults including ischemia, oxidative stress and cytokine injury that cause a reduction in the recovered viable islet mass. A novel approach was designed in which streptozotocin (STZ)-damaged rat pancreatic islets (rPIs) were indirectly cocultured with rat bone marrow-derived mesenchymal stem cells (rBM-MSCs) to maintain survival of the cultured rPIs. The results indicated that islets cocultured with rBM-MSCs secreted an increased level of insulin after 14 days, whereas non-cocultured islets gradually deteriorated and cell death occurred. The cocultivation of rBM-MSCs with islets and STZ-damaged islets showed the expression of IL6 and transforming growth factor-β1 in the culture medium, besides the expression of the antiapoptotic genes (Mapkapk2, Tnip1 and Bcl3), implying the cytoprotective, anti-inflammatory and antiapoptotic effects of rBM-SCs through paracrine actions.     

Preparation of suspensions of pancreatic islet cells: a comparison of methods

A comparative study for preparation of cell suspensions from pancreatic islets has been performed using mechanical or enzymatic dissociation with proteolytic enzymes such as trypsin, dispase, and pronase. Treatment of isolated pancreatic islets from neonatal rats with these enzymes proved to be superior to a mechanical dissociation method. The enzymatic dissociation was performed by fractionated treatment of pancreatic islets with low concentration of enzymes in Hanks' solution for 2-3 min at room temperature. With the exception of trypsin the percentage of single cells was consistently higher with dispase and pronase treatment, being 83-92%. Cell viability (dye exclusion) was more than 90%. Mechanical disintegration of pancreatic islets resulted in a low yield of single cells, and cell viability was considerably reduced in comparison with the enzymatic methods. Labeling of islet cells with Na2 51CrO4 and measurement of the basal 51Cr-release demonstrated superior membrane preservation after pronase or dispase treatment. Islet cells isolated either by fractionated dispase or pronase treatment were found to be well preserved and very suitable for the detection of circulating cell surface antibodies and their cytotoxic effects to islet cells.     

Rapid isolation of pancreatic islets from collagenase digested pancreas by sedimentation through Percol at unit gravity.

A Percoll solution with a density of 1.045 g/ml was used to separate pancreatic islets and exocrine tissue from collagenase-digested human and $\text{ob/ob}$ mouse pancreases by sedimentation at unit gravity. Most exocrine tissue from the mouse was found to range in density from 1.015 to 1.045 g/ml whereas the denser islets lay in a narrower range of 1.065–1.070 g/ml. Up to 400 islets were obtained from each mouse pancreas and 140 islets from 4 g of human pancreas; the isolated islets being essentially free from contamination with exocrine tissue. Glucose-stimulated insulin release was the same whether the mouse islets were isolated with or without Percoll. The simplicity of the method makes it suitable for large-scale islet isolation, a feature of potential importance for the treatment of diabetes by islet transplantation.     

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 ¹. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established ². Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Lepr^db (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.