Acetate and Butyrate Improve ß-cell Metabolism and Mitochondrial Respiration under Oxidative Stress.

Islet dysfunction mediated by oxidative and mitochondrial stress contributes to the development of type 1 and 2 diabetes. Acetate and butyrate, produced by gut microbiota via fermentation, have been shown to protect against oxidative and mitochondrial stress in many cell types, but their effect on pancreatic ß-cell metabolism has not been studied. Here, human islets and the mouse insulinoma cell line MIN6 were pre-incubated with 1, 2, and 4 mM of acetate or butyrate with and without exposure to the apoptosis inducer and metabolic stressor streptozotocin (STZ). Both short-chain fatty acids (SCFAs) enhanced the viability of islets and ß-cells, but the beneficial effects were more pronounced in the presence of STZ. Both SCFAs prevented STZ-induced cell apoptosis, viability reduction, mitochondrial dysfunction, and the overproduction of reactive oxygen species (ROS) and nitric oxide (NO) at a concentration of 1 mM but not at higher concentrations. These rescue effects of SCFAs were accompanied by preventing reduction of the mitochondrial fusion genes MFN, MFN2, and OPA1. In addition, elevation of the fission genes DRP1 and FIS1 during STZ exposure was prevented. Acetate showed more efficiency in enhancing metabolism and inhibiting ROS, while butyrate had less effect but was stronger in inhibiting the SCFA receptor GPR41 and NO generation. Our data suggest that SCFAs play an essential role in supporting ß-cell metabolism and promoting survival under stressful conditions. It therewith provides a novel mechanism by which enhanced dietary fiber intake contributes to the reduction of Western diseases such as diabetes.

Extracellular matrix inclusion in immunoisolating alginate-based microcapsules promotes longevity, reduces fibrosis, and supports function of islet allografts in vivo.

Immunoisolation of pancreatic-islets in alginate-microcapsules is applied to treat diabetes. However, long-term islet function is limited, which might be due to damaged and lack of contact with pancreatic extracellular matrix (ECM) components. Herein we investigated the impact of collagen IV combined with laminin sequences, either RGD, LRE, or PDSGR, on graft-survival of microencapsulated bioluminescent islets in vivo. Collagen IV with RGD had the most pronounced effect. It enhanced after 8-week implantation in immune-incompetent mice the bioluminescence of allogeneic islets by 3.2-fold, oxygen consumption rate by 14.3-fold and glucose-induced insulin release by 9.6-fold. Transcriptomics demonstrated that ECM enhanced canonical pathways involving insulin-secretion and that it suppressed pathways related to inflammation and hypoxic stress. Also, 5.8-fold fewer capsules were affected by fibrosis. In a subsequent longevity study in immune-competent mice, microencapsulated allografts containing collagen IV and RGD had a 2.4-fold higher functionality in the first week after implantation and remained at least 2.1-fold higher during the study. Islets in microcapsules containing collagen IV and RGD survived 211 ± 24.1 days while controls survived 125 ± 19.7 days. Our findings provide in vivo evidence for the efficacy of supplementing immunoisolating devices with specific ECM components to enhance functionality and longevity of islet-grafts in vivo. STATEMENT OF SIGNIFICANCE: Limitations in duration of survival of immunoisolated pancreatic islet grafts is a major obstacle for application of the technology to treat diabetes. Accumulating evidence supports that incorporation of extracellular matrix (ECM) molecules in the capsules enhances longevity of pancreatic islets. After selection of the most efficacious laminin sequence in vitro, we show in vivo that inclusion of collagen IV and RGD in alginate-based microcapsules enhances survival, insulin secretion function, and mitochondrial function. It also suppresses fibrosis by lowering proinflammatory cytokines secretion. Moreover, transcriptomic analysis shows that ECM-inclusion promotes insulin-secretion related pathways and attenuates inflammation and hypoxic stress related pathways in islets. We show that inclusion of ECM in immunoisolating devices is a promising strategy to promote long-term survival of islet-grafts.

Applying Immunomodulation to Promote Longevity of Immunoisolated Pancreatic Islet Grafts.

Islet transplantation is a promising therapy for insulin-dependent diabetes, but large-scale application is hampered by the lack of a consistent source of insulin-producing cells and need for lifelong administration of immunosuppressive drugs, which are associated with severe side effects. To avoid chronic immunosuppression, islet grafts can be enveloped in immunoisolating polymeric membranes. These immunoisolating polymeric membranes protect islet grafts from cell-mediated rejection while allowing diffusion of oxygen, nutrients, and insulin. Although clinical trials have shown the safety and feasibility of encapsulated islets to control glucose homeostasis, the strategy does up till now not support long-term graft survival. This partly can be explained by a significant loss of insulin-producing cells in the immediate period after implantation. The loss can be prevented by combining immunoisolation with immunomodulation, such as combined administration of immunomodulating cytokines or coencapsulation of immunomodulating cell types such as regulatory T cells, mesenchymal stem cells, or Sertoli cells. Also, administration of specific antibodies or apoptotic donor leucocytes is considered to create a tolerant microenvironment around immunoisolated grafts. In this review, we describe the outcomes and limitations of these approaches, as well as the recent progress in immunoisolating devices. Impact statement Immunoisolation by enveloping islets in semipermeable membranes allows for successful transplantation of islet grafts in the absence of chronic immunosuppression, but the duration of graft survival is still not permanent. The reasons for long-term final graft failure is not fully understood, but combining immunoisolation with immunomodulation of tissues or host immune system has been proposed to enhance the longevity of grafts. This article reviews the recent progress and challenges of immunoisolation, as well as the benefits and feasibility of combining encapsulation approaches with immunomodulation to promote longevity of encapsulated grafts.

Quantitative Cell Subset Analysis Using Antibody Microarrays.

The expression patterns of surface antigens are associated with the differentiation status and functional characteristics of mammalian cells. To analyze the surface antigen expression pattern in a high-throughput manner, antibody microarrays have been developed by several groups, including ours. This analysis can be performed using cell-binding assays on microarrays; moreover, this approach has advantages over conventional flow cytometry (FCM). Unlike FCM, the microarray-based method cannot evaluate the concurrent expression of more than two surface antigens on a single cell, and therefore, it cannot be used for cell subset analysis. To overcome this drawback, we prepared an antibody microarray with spots presenting co-immobilized multiple antibodies together with spots presenting each antibody separately. The co-immobilized spots are expected to be reactive for every surface antigen specific to the co-immobilized antibodies. In addition, the concept of an algebra of sets is incorporated into the derivation of quantitative data regarding cell subsets. Here, taking cell subsets with respect to two surface antigens as the simplest example, antibody microarrays were prepared and initially subjected to validation studies to verify the accuracy of cell-binding assays. Quantitative subset analysis was performed using antibody microarrays prepared using the anti-CD13 and anti-CD49f antibodies. For model populations that consisted of discrete subsets, THP-1, HL-60, CCRF-CEM, and Ramos cell lines were used because they were found by FCM to have a singular phenotype, that is, CD13+CD49f+, CD13+CD49f-, CD13-CD49f+, and CD13-CD49f-, respectively. Five populations were prepared by mixing these cells at various ratios and analyzed for their subsets using microarrays. The results showed that the experimentally determined abundance ratios of the four model subsets were in good agreement with the predetermined abundance ratios, which provided the proof of principle for the new method in the quantitative subset analysis.

In vivovascularization and islet function in a microwell device for pancreatic islet transplantation.

Islet encapsulation in membrane-based devices could allow for transplantation of donor islet tissue in the absence of immunosuppression. To achieve long-term survival of islets, the device should allow rapid exchange of essential nutrients and be vascularized to guarantee continued support of islet function. Recently, we have proposed a membrane-based macroencapsulation device consisting of a microwell membrane for islet separation covered by a micropatterned membrane lid. The device can prevent islet aggregation and support functional islet survivalin vitro. Here, based on previous modeling studies, we develop an improved device with smaller microwell dimensions, decreased spacing between the microwells and reduced membrane thickness and investigate its performancein vitroandin vivo. This improved device allows for encapsulating higher islet numbers without islet aggregation and by applying anin vivoimaging system we demonstrate very good perfusion of the device when implanted intraperitoneally in mice. Besides, when it is implanted subcutaneously in mice, islet viability is maintained and a vascular network in close proximity to the device is developed. All these important findings demonstrate the potential of this device for islet transplantation.

Toll-like receptor 2-modulating pectin-polymers in alginate-based microcapsules attenuate immune responses and support islet-xenograft survival.

Encapsulation of pancreatic islets in alginate-microcapsules is used to reduce or avoid the application of life-long immunosuppression in preventing rejection. Long-term graft function, however, is limited due to varying degrees of host tissue responses against the capsules. Major graft-longevity limiting responses include inflammatory responses provoked by biomaterials and islet-derived danger-associated molecular patterns (DAMPs). This paper reports on a novel strategy for engineering alginate microcapsules presenting immunomodulatory polymer pectin with varying degrees of methyl-esterification (DM) to reduce these host tissue responses. DM18-pectin/alginate microcapsules show a significant decrease of DAMP-induced Toll-Like Receptor-2 mediated immune activation in vitro, and reduce peri-capsular fibrosis in vivo in mice compared to higher DM-pectin/alginate microcapsules and conventional alginate microcapsules. By testing efficacy of DM18-pectin/alginate microcapsules in vivo, we demonstrate that low-DM pectin support long-term survival of xenotransplanted rat islets in diabetic mice. This study provides a novel strategy to attenuate host responses by creating immunomodulatory capsule surfaces that attenuate activation of specific pro-inflammatory immune receptors locally at the transplantation site.

Low methyl-esterified pectin protects pancreatic ß-cells against diabetes-induced oxidative and inflammatory stress via galectin-3.

Insufficient intake of dietary fibers in Western societies is considered a major contributing factor in the high incidence rates of diabetes. The dietary fiber pectin has been suggested to be beneficial for management of both Diabetes Type 1 and Type 2, but mechanisms and effects of pectin on insulin producing pancreatic ß-cells are unknown. Our study aimed to determine the effects of lemon pectins with different degree of methyl-esterification (DM) on ß-cells under oxidative (streptozotocin) and inflammatory (cytokine) stress and to elucidate the underlying rescuing mechanisms, including effects on galectin-3. We found that specific pectins had rescuing effects on toxin and cytokine induced stress on ß-cells but effects depended on the pectin concentration and DM-value. Protection was more pronounced with low DM5 pectin and was enhanced with higher pectin-concentrations. Our findings show that specific pectins might prevent diabetes by making insulin producing ß-cells less susceptible for stress.

Bioabsorbable device to prepare subcutaneous pockets for islet transplantation.

Small invasive transplantation of islets and long maintenance of the islet graft without immunosuppression has been studied for the treatment of type 1 diabetes. Clinically, subcutaneous pockets surrounding vascular-rich tissue are prepared for islet transplantation using a device made of the materials. Here, gelatin sheets were implanted into two dorsal subcutaneous sites in diabetic ACI rats, and a mixture of bFGF and sodium hyaluronate solution was injected around the gelatin sheets. A total of 1500 islets isolated from F344 rats were transplanted into each of the pockets 7 days after injection of the bFGF mixture. Nine of 10 diabetic ACI rats with allogeneic islet graft demonstrated long-term normoglycemia without administration of immunosuppressant. Gelatin sheets almost disappeared 67 days after implantation. Thus, subcutaneous immune-tolerant sites can be prepared using gelatin sheets and a sodium hyaluronate-bFGF mixture. Allogeneic islets transplanted into the sites can survive and control blood glucose levels for a long period, even without immunosuppression. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1107-1112, 2019.

Preparation of Immunotolerant Space Under the Skin and Transplantation of Islets in the Space.

IMPACT STATEMENT: Although transplantation of islets of Langerhans has been accepted as a fundamental treatment for insulin-dependent diabetes mellitus (IDDM), several problems are still remaining in order that it becomes the standard medical treatment of IDDM patients. In this study, using diabetic rat models, a subcutaneous space surrounded with highly vascularized granulomatous tissue was formed by agarose-bFGF rod implantation. Allogeneic islets transplanted into the space could survive and release insulin for a long period under no immunosuppressive medication. In conjunction with sufficient supply of islets from iPS/ES cells, our method can make islet transplantation the standard medical treatment of IDDM patients.

Long-term Functioning of Allogeneic Islets in Subcutaneous Tissue Pretreated With a Novel Cyclic Peptide Without Immunosuppressive Medication.

BACKGROUND: There exists a need for a minimally invasive method of islet transplantation without immunosuppressive drugs for the treatment of type 1 diabetes. METHODS: In diabetic August Copenhagen Irish rats, an agarose rod containing the cyclic oligopeptide SEK-1005 (agarose-SEK rod) was implanted at 2 dorsal subcutaneous sites. Then these rods were removed, and 1500 islets of Langerhans isolated from Fischer 344 rats were transplanted into each of the pockets. RESULTS: Ten days after implantation of agarose-SEK rods, vascularized pockets were present. Nonfasting blood glucose levels confirmed long-term survival of the allogeneic islet grafts, without immunosuppressive therapy, in 8 of 10 recipients. Flow cytometry and gene expression analyses were performed to investigate the mechanisms underlying graft acceptance. Agarose-SEK rod implantation led to the formation of granulomatous tissue containing regulatory T cells that suppressed immune reactions against the allogeneic islet grafts. CONCLUSIONS: These results indicate that the use of an agarose-SEK rod to prevascularize a subcutaneous site may be a useful method for achieving successful allogeneic islet transplantation without immunosuppression.

Coaggregates of Regulatory T Cells and Islet Cells Allow Long-term Graft Survival in Liver Without Immunosuppression.

BACKGROUND: Transplantation of islets of Langerhans (islets) has been investigated in the clinic to treat patients with insulin-dependent diabetes mellitus. Islet grafts have been maintained by administering immunosuppressive drugs, which can lead to complications in the long term. Alternatives to immunosuppressive therapy are eagerly desired. In this study, we examined the transplantation of coaggregates of CD4CD25 regulatory T (Treg) cells. METHODS: Coaggregates of Treg cells from C57BL/6 mice and islet cells from BALB/c mice were prepared on agarose hydrogel with small round-bottomed wells. Four hundred coaggregates were transplanted into the livers of streptozotocin-induced diabetic C57BL/6 mice without systemic immunosuppression. RESULTS: The Treg cells and islet cells were distributed randomly in the coaggregates. When 400 coaggregates were transplanted into 9 C57BL/6 mice via the portal vein, 6 of the 9 recipients demonstrated blood glucose less than 250 mg/dL for more than 100 days. A number of insulin-positive cells were observed in the livers at 120 days after transplantation. CONCLUSIONS: The Treg cells and islet cells were distributed randomly in the coaggregates. After intraportal transplantation of the coaggregates, Treg cells in the aggregates enabled the long-term survival of allogeneic islet cell grafts in the liver without the use of immunosuppressive drugs.

Interaction between cells and poly(ethylene glycol)-lipid conjugates.

Eight types of poly(ethylene glycol)-lipid(PEG-lipids) carrying different lipid tails were synthesized. These PEG-lipids were labeled with fluorescein isothiocyanate (FITC-PEG-lipids) to examine their interaction with cells and to quantitatively determine amounts of PEG-lipids bound on the cell surface. FITC-PEG-lipids spontaneously anchored to the cell membrane within 15 min without loss of cell viability. The type of lipid had very little effect on the anchoring rates, while an increase in the hydrophobicity of the lipid portion of the PEG-lipids slowed their dissociation rates. Densities of FITC-PEG-lipids on the cell surface ranged from 1 × 10(-3) to 1 × 10(-2)molecules/nm(2), depending on the kinds of lipids employed. The relationship between the stability of the lipids on the cell membrane and the hydrophobicity of the lipid moieties will give a basis for the selection of a hydrophobic moiety in PEG-lipid conjugates for use in specific applications.