GABA protects pancreatic beta cells against apoptosis by increasing SIRT1 expression and activity.

We have previously shown that GABA protects pancreatic islet cells against apoptosis and exerts anti-inflammatory effects. Notably, GABA inhibited the activation of NF-κB in both islet cells and lymphocytes. NF-κB activation is detrimental to beta cells by promoting apoptosis. However, the mechanisms by which GABA mediates these effects are unknown. Because the above-mentioned effects mimic the activity of sirtuin 1 (SIRT1) in beta cells, we investigated whether it is involved. SIRT1 is an NAD(+)-dependent deacetylase that enhances insulin secretion, and counteracts inflammatory signals in beta cells. We found that the incubation of a clonal beta-cell line (rat INS-1) with GABA increased the expression of SIRT1, as did GABA receptor agonists acting on either type A or B receptors. NAD(+) (an essential cofactor of SIRT1) was also increased. GABA augmented SIRT1 enzymatic activity, which resulted in deacetylation of the p65 component of NF-κB, and this is known to interfere with the activation this pathway. GABA increased insulin production and reduced drug-induced apoptosis, and these actions were reversed by SIRT1 inhibitors. We examined whether SIRT1 is similarly induced in newly isolated human islet cells. Indeed, GABA increased both NAD(+) and SIRT1 (but not sirtuins 2, 3 and 6). It protected human islet cells against spontaneous apoptosis in culture, and this was negated by a SIRT1 inhibitor. Thus, our findings suggest that major beneficial effects of GABA on beta cells are due to increased SIRT1 and NAD(+), and point to a new pathway for diabetes therapy.

An in vitro Perfused Macroencapsulation Device to Study Hemocompatibility and Survival of Islet-Like Cell Clusters.

Transplantation of hydrogel-encapsulated pancreatic islets is a promising long-term treatment for type 1 diabetes that restores blood glucose regulation while providing graft immunoprotection. Most human-scale islet encapsulation devices that rely solely on diffusion fail to provide sufficient surface area to meet islet oxygen demands. Perfused macroencapsulation devices use blood flow to mitigate oxygen limitations but increase the complexity of blood-device interactions. Here we describe a human-scale in vitro perfusion system to study hemocompatibility and performance of islet-like cell clusters (ILCs) in alginate hydrogel. A cylindrical perfusion device was designed for multi-day culture without leakage, contamination, or flow occlusion. Rat blood perfusion was assessed for prothrombin time and international normalized ratio and demonstrated no significant change in clotting time. Ex vivo perfusion performed with rats showed patency of the device for over 100 min using Doppler ultrasound imaging. PET-CT imaging of the device successfully visualized metabolically active mouse insulinoma 6 ILCs. ILCs cultured for 7 days under static conditions exhibited abnormal morphology and increased activated caspase-3 staining when compared with the perfused device. These findings reinforce the need for convective transport in macroencapsulation strategies and offer a robust and versatile in vitro system to better inform preclinical design.

Monitoring the survival of islet transplants by MRI using a novel technique for their automated detection and quantification.

OBJECT: There is a clinical need to be able to assess graft loss of transplanted pancreatic islets (PI) non-invasively with clear-cut quantification of islet survival. We tracked transplanted PI in diabetic mice during the early post-transplant period by magnetic resonance imaging (MRI) and quantified the islet loss using automatic segmentation technique. MATERIALS AND METHODS: Magnetically labeled islet iso-, allo- and xenografts were injected into the right liver lobes. Animals underwent MRI scanning during 14 days after PI transplantation. MR images were processed using custom-made software, which automatically detects hypointense regions representing PI. It is based on morphological top-hat and bottom-hat transforms. RESULTS: Manually and automatically detected areas, corresponding to PI, differed by 4% in phantoms. Signal loss regions due to PI decreased comparably in all groups during the first week post transplant. Throughout the second week post-transplant, the signal loss area continued in a steep decline in case of allografts and xenografts, whereas the decline in case of isografts slowed down. CONCLUSION: Automatic segmentation allows for the more reproducible, objective assessment of transplanted PI. Quantification confirms the assumption that a significant number of islets are destroyed in the first week following transplantation irrespective of allografts, xenografts or isografts.

Oxygen Perfusion (Persufflation) of Human Pancreata Enhances Insulin Secretion and Attenuates Islet Proinflammatory Signaling.

BACKGROUND: All human islets used in research and for the clinical treatment of diabetes are subject to ischemic damage during pancreas procurement, preservation, and islet isolation. A major factor influencing islet function is exposure of pancreata to cold ischemia during unavoidable windows of preservation by static cold storage (SCS). Improved preservation methods may prevent this functional deterioration. In the present study, we investigated whether pancreas preservation by gaseous oxygen perfusion (persufflation) better preserved islet function versus SCS. METHODS: Human pancreata were preserved by SCS or by persufflation in combination with SCS. Islets were subsequently isolated, and preparations in each group matched for SCS or total preservation time were compared using dynamic glucose-stimulated insulin secretion as a measure of ß-cell function and RNA sequencing to elucidate transcriptomic changes. RESULTS: Persufflated pancreata had reduced SCS time, which resulted in islets with higher glucose-stimulated insulin secretion compared to islets from SCS only pancreata. RNA sequencing of islets from persufflated pancreata identified reduced inflammatory and greater metabolic gene expression, consistent with expectations of reducing cold ischemic exposure. Portions of these transcriptional responses were not associated with time spent in SCS and were attributable to pancreatic reoxygenation. Furthermore, persufflation extended the total preservation time by 50% without any detectable decline in islet function or viability. CONCLUSIONS: These data demonstrate that pancreas preservation by persufflation rather than SCS before islet isolation reduces inflammatory responses and promotes metabolic pathways in human islets, which results in improved ß cell function.

Isolating human islets of Langerhans causes loss of decay accelerating factor (CD55) on beta-cells.

It has previously been reported that human decay accelerating factor (DAF; CD55) is not expressed on cells isolated from human islets. We have investigated if this absence is caused by the islet isolation procedure and/or the single cell isolation technique. We focused on loss of DAF expression on beta-cells within the intact islet and on isolated individual beta-cells. We established that DAF was expressed in islets and on beta-cells prior to isolation by in situ analysis in the intact pancreas. In situ immunohistochemistry (IHC) was used to examine DAF expression on human pancreatic islets and isolated islets. A reverse transcriptase-polymerase chain reaction (RT-PCR) specific for human DAF mRNA was developed to measure mRNA levels in situ in islets within the intact pancreas, isolated islets, and purified beta-cells. beta-Cells were purified by fluorescence-activated cell sorting. DAF protein expression on these purified cells was measured using flow cytometry. Expression of DAF protein was present on the islets, including beta-cells within the human pancreas; however, comparative data from IHC and flow cytometry revealed the absence of DAF protein on beta-cells in both isolated islets and single cell preparations. Furthermore, compared to mRNA levels detected by in situ RT-PCR in the intact pancreas and in human HEK 293 cells, isolated islets, and purified human beta-cells showed downregulation of DAF mRNA. mRNA was detectable in both of these preparations by RT-PCR; levels were lower following both the islet isolation process (53%) and single cell preparation (a further 62%) compared to HEK 293 controls. Human islet allotransplantation might be more successful if either de novo transfer of DAF onto the isolated islets or novel techniques for islet isolation preserving DAF could be developed.

Overcoming barriers to facilitate the regulation of multi-centre regenerative medicine clinical trials.

In the context of regenerative medicine and cellular therapies, the treatment under study often targets a less common disease or condition for which recruitment of a large number of research participants at any given site is challenging, if not impossible. One way to overcome this challenge is with a multi-centre clinical trial. This manuscript first aims to briefly outline the existing ethical, legal and social implications as well as the regulatory frameworks associated with multi-centre regenerative medicine clinical trials. Second, it considers the regulatory limitations and barriers surrounding the initiation of such trials in Canada, the USA and Europe. Third, it concludes with a set of recommendations for facilitating multi-centre clinical trials, at both national and international levels.

Immune Response to Extracellular Vesicles From Human Islets of Langerhans in Patients With Type 1 Diabetes.

The autoimmune response that characterizes type 1 diabetes (T1D) has no clear cause. Extracellular vesicles (EVs) play an important role in triggering the immune response in other contexts. Here, we propose a model by which EVs isolated from human islets stimulate proinflammatory immune responses and lead to peripheral blood mononuclear cell (PBMC) activation. We show that human islet EVs are internalized by monocytes and B cells and lead to an increase in T-helper 1, 2, and 17 cytokine expression, as well as T and B cell proliferation. Importantly, we demonstrate memory T and B cell activation by EVs selectively in PBMCs of patients with T1D. Additionally, human islet EVs induce an increase in antibodies against glutamic acid decarboxylase 65 (GAD65) in T1D PBMCs. Furthermore, pretreatment of T1D PBMCs with ibrutinib, an inhibitor of Bruton tyrosine kinase, dampens EV-induced memory B cell activation and GAD65 antibody production. Collectively, our findings indicate a role for human islet EVs in mediating activation of B and T cells and GAD65 autoantibody production.

Imaging islets labeled with magnetic nanoparticles at 1.5 Tesla.

We have developed a magnetic resonance imaging (MRI) technique for imaging Feridex (superparamagnetic iron oxide [SPIO])-labeled islets of Langerhans using a standard clinical 1.5-Tesla (T) scanner and employing steady-state acquisition imaging sequence (3DFIESTA). Both porcine and rat islets were labeled with SPIO by a transfection technique using a combination of poly-l-lysine and electroporation. Electron microscopy demonstrated presence of SPIO particles within the individual islet cells, including beta-cells and particles trapped between cell membranes. Our labeling method produced a transfection rate of 860 pg to 3.4 ng iron per islet, dependent on the size of the islet. The labeling procedure did not disrupt either the function or viability of the islets. In vitro 3DFIESTA magnetic resonance images of single-labeled islets corresponded with their optical images. In vivo T2*-weighted scan using 1.5 T detected as few as 200 SPIO-labeled islets transplanted under rat kidney capsule, which correlated with immunohistochemistry of the transplant for insulin and iron. Ex vivo 3DFIESTA images of kidneys containing 200, 800 or 2,000 SPIO-labeled islet isografts showed good correlation between signal loss and increasing numbers of islets. These data provide evidence that islets can be labeled with SPIO and imaged using clinically available 1.5- T MRI.

Presence of diabetes autoantigens in extracellular vesicles derived from human islets.

Beta-cell (ß-cell) injury is the hallmark of autoimmune diabetes. However, the mechanisms by which autoreactive responses are generated in susceptible individuals are not well understood. Extracellular vesicles (EV) are produced by mammalian cells under normal and stressed physiological states. They are an important part of cellular communication, and may serve a role in antigen processing and presentation. We hypothesized that isolated human islets in culture produce EV that contain diabetes autoantigens (DAA) from these otherwise normal, non-diabetic donors. Here we report the caspase-independent production of EV by human islets in culture, and the characterization of DAA glutamic acid decarboxylase 65 (GAD65) and zinc transporter 8 (ZnT8), as well as the ß-cell resident glucose transporter 2 (Glut2), present within the EV.

GABA promotes human ß-cell proliferation and modulates glucose homeostasis.

γ-Aminobutyric acid (GABA) exerts protective and regenerative effects on mouse islet ß-cells. However, in humans it is unknown whether it can increase ß-cell mass and improve glucose homeostasis. To address this question, we transplanted a suboptimal mass of human islets into immunodeficient NOD-scid-γ mice with streptozotocin-induced diabetes. GABA treatment increased grafted ß-cell proliferation, while decreasing apoptosis, leading to enhanced ß-cell mass. This was associated with increased circulating human insulin and reduced glucagon levels. Importantly, GABA administration lowered blood glucose levels and improved glucose excursion rates. We investigated GABA receptor expression and signaling mechanisms. In human islets, GABA activated a calcium-dependent signaling pathway through both GABA A receptor and GABA B receptor. This activated the phosphatidylinositol 3-kinase-Akt and CREB-IRS-2 signaling pathways that convey GABA signals responsible for ß-cell proliferation and survival. Our findings suggest that GABA regulates human ß-cell mass and may be beneficial for the treatment of diabetes or improvement of islet transplantation.

GABA protects human islet cells against the deleterious effects of immunosuppressive drugs and exerts immunoinhibitory effects alone.

BACKGROUND: We recently found that γ-aminobutyric acid (GABA) protects mouse islet ß cells. It prevented autoimmune type 1 diabetes in mice, induced islet ß-cell regeneration, and exerted immunoinhibitory effects. However, it is not known whether GABA would be equally active on human islet and immune cells. METHODS: In vitro culture of human islets and immune cells with or without GABA and immunosuppressive drugs. In vitro analysis of apoptosis, proliferation, nuclear factor (NF)-κB activation, calcium signaling, and insulin secretion. RESULTS: GABA reduced human islet cell apoptosis in culture, such that the yield of live cells was approximately tripled after 1 week, and it stimulated insulin secretion. It protected against the deleterious effects of rapamycin, tacrolimus, and mycophenolate mofetil. In human immune cells, GABA had inhibitory effects similar to mouse cells, such as suppressed anti-CD3-stimulated T-cell proliferation, in a GABA type A receptor-dependent fashion. The immunosuppressive mechanisms have been unclear, but we found that GABA blocked calcium influx, which is a key activation signal. GABA also suppressed NF-κB activation in both human islet cells and immune cells. We found that it could be combined with rapamycin to increase its suppressive effects. CONCLUSIONS: GABA improved human islet cell survival and had suppressive effects on human immune cells. It inhibited canonical NF-κB activation in both islet and immune cells. This is important because activation of this pathway is detrimental to islet cells and likely promotes damaging autoimmunity and alloreactivity against transplanted islets. These findings suggest that GABA might find applications in clinical islet transplantation.

Analysis of beta-cell gene expression reveals inflammatory signaling and evidence of dedifferentiation following human islet isolation and culture.

The stresses encountered during islet isolation and culture may have deleterious effects on beta-cell physiology. However, the biological response of human islet cells to isolation remains poorly characterized. A better understanding of the network of signaling pathways induced by islet isolation and culturing may lead to strategies aimed at improving islet graft survival and function. Laser capture microdissection (LCM) was used to extract beta-cell RNA from 1) intact pancreatic islets, 2) freshly isolated islets, 3) islets cultured for 3 days, and changes in gene expression were examined by microarray analysis. We identified a strong inflammatory response induced by islet isolation that continues during in-vitro culture manifested by upregulation of several cytokines and cytokine-receptors. The most highly upregulated gene, interleukin-8 (IL-8), was induced by 3.6-fold following islet isolation and 56-fold after 3 days in culture. Immunofluorescence studies showed that the majority of IL-8 was produced by beta-cells themselves. We also observed that several pancreas-specific transcription factors were down-regulated in cultured islets. Concordantly, several pancreatic progenitor cell-specific transcription factors like SOX4, SOX9, and ID2 were upregulated in cultured islets, suggesting progressive transformation of mature beta-cell phenotype toward an immature endocrine cell phenotype. Our findings suggest islet isolation and culture induces an inflammatory response and loss of the mature endocrine cell phenotype. A better understanding of the signals required to maintain a mature beta-cell phenotype may help improve the efficacy of islet transplantation.

The islet size to oxygen consumption ratio reliably predicts reversal of diabetes posttransplant.

ß-Cell replacement therapy by either whole-organ pancreas or islets of Langerhans transplantation can restore carbohydrate control to diabetic patients and reduces complications associated with the disease. One of the variables inherent in islet transplantation is the isolation of functional islets from donor pancreata. Islet isolations fail to consistently produce good-quality functional islets. A rapid pretransplant assay to determine posttransplant function of islets would be an invaluable tool. We have tested the novel hypothesis that modified oxygen consumption rates (OCR), standardized to DNA quantity (nmol/min-mg DNA), would serve as a pretransplant assessment of the metabolic potency of the islets postisolation. This study compares the ability of current in vitro assays to predict in vivo restoration of normoglycemia in a diabetic nude mouse posttransplantation of adult pig islets. There is known to be a diversity of islet sizes within each preparation. This parameter has not heretofore been effectively considered a critical factor in islet engraftment. Our results suggest a surprising finding that islet size influences the probability of restoring carbohydrate control. Based on this observation, we thus developed a novel predictor of islet graft function that combines the effects of both islet OCR and size. When OCR was divided by the islet index (size), a highly significant predictor of graft function was established (p = 0.0002, n = 75). Furthermore, when OCR/islet index values exceeded 70.0 nmol/min-mg DNA/islet index, an effective threshold of diabetes reversal was observed. This assay can be performed with as few as 1,000 islet equivalents (IEQ) and conducted in less than 60 min. Our data suggest that, using this novel method to assess islet cell function prior to transplantation, OCR/islet index thresholds provide a valuable tool in identifying which islet preparations are most likely to restore glycemic control posttransplant.

Nuclear targeting of stanniocalcin to mammary gland alveolar cells during pregnancy and lactation.

In most mammalian tissues, the stanniocalcin-1 gene (STC-1) produces a 50-kDa polypeptide hormone known as STC50. Within the ovaries, however, the STC-1 gene generates three higher-molecular-mass variants known as big STC. Big STC is targeted locally to corpus luteal cells to block progesterone release. During pregnancy and lactation, however, ovarian big STC production increases markedly, and the hormone is released into the serum. During lactation, this increase in hormone production is dependent on a suckling stimulus, suggesting that ovarian big STC may have regulatory effects on the lactating mammary gland. In this report, we have addressed this possibility. Our results revealed that virgin mammary tissue contained large numbers of membrane- and mitochondrial-associated STC receptors. However, as pregnancy progressed into lactation, there was a decline in receptor densities on both organelles and a corresponding rise in nuclear receptor density, most of which were on milk-producing, alveolar cells. This was accompanied by nuclear sequestration of the ligand. Sequestered STC resolved as one approximately 135-kDa band in the native state and therefore had the appearance of a big STC variant. However, chemical reduction collapsed this one band into six closely spaced, lower-molecular-mass species (28-41 kDa). Mammary gland STC production also underwent a dramatic shift during pregnancy and lactation. High levels of STC gene expression were observed in mammary tissue from virgin and pregnant rats. However, gene expression then fell to nearly undetectable levels during lactation, coinciding with the rise in nuclear targeting. These findings have thus shown that the mammary glands are indeed targeted by STC, even in the virgin state. They have further shown that there are marked changes in this targeting pathway during pregnancy and lactation, accompanied by a switch in ligand source (endogenous to exogenous). They also represent the first example of nuclear targeting by STC.

Characterization of mammalian stanniocalcin receptors. Mitochondrial targeting of ligand and receptor for regulation of cellular metabolism.

The polypeptide hormone stanniocalcin (STC) is widely expressed in mammalian tissues. STC acts locally in kidney and gut to modulate calcium and phosphate excretion, and its overexpression in mice results in high serum phosphate, dwarfism, and increased metabolic rate. STC has also been linked to cancer, pregnancy, lactation, angiogenesis, organogenesis, cerebral ischemia, and hypertonic stress. In this report we have characterized the STC receptor and the functional targeting of ligand and receptor to mitochondria. For receptor binding analysis, a stanniocalcin-alkaline phosphatase fusion protein was engineered. Subsequent binding assays using the fusion protein indicated that kidney and liver contained the highest number of binding sites with affinities of 0.8 and 0.25 nm, respectively. Intriguingly, purified mitochondria from both tissues yielded similar high affinity binding sites. Fractionation analysis revealed that the majority of binding sites were localized to the inner mitochondrial membrane. In further studies, we characterized the time course of STC-alkaline phosphatase fusion protein sequestration by intact mitochondria. In situ ligand binding also revealed discrete, displaceable binding to plasma membranes and mitochondria of nephron cells and liver hepatocytes. The existence of mitochondrial receptors prompted a similar search for the ligand. Immunogold electron microscopy revealed that STC was preferentially concentrated in the mitochondria of all nephron segments targeted by STC. Subcellular fractionation revealed that >90% of cellular STC immunoreactivity was mitochondrial, confined to the inner matrix, and similar in size to recombinant STC (50 kDa). In functional studies, recombinant STC had concentration-dependent stimulatory effects on electron transfer by sub-mitochondrial particles. Collectively the evidence implies a role for STC in cell metabolism.