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Keywords:

  • Early loss of transplanted islets;
  • hypoxia;
  • islet transplantation;
  • Na+/Ca2+ exchanger

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. References
  10. Supporting Information

Pancreatic islet transplantation is an attractive therapy for the treatment of insulin-dependent diabetes mellitus. However, the low efficiency of this procedure necessitating sequential transplantations of islets with the use of 2–3 donors for a single recipient, mainly due to the early loss of transplanted islets, hampers its clinical application. Previously, we have shown in mice that a large amount of HMGB1 is released from islets soon after their transplantation and that this triggers innate immune rejection with activation of DC, NKT cells and neutrophils to produce IFN-γ, ultimately leading to the early loss of transplanted islets. Thus, HMGB1 release plays an initial pivotal role in this process; however, its mechanism remains unclear. Here we demonstrate that release of HMGB1 from transplanted islets is due to hypoxic damage resulting from Ca2+ influx into β cells through the Na+/Ca2+ exchanger (NCX). Moreover, the hypoxia-induced β cell damage was prevented by pretreatment with an NCX-specific inhibitor prior to transplantation, resulting in protection and long-term survival of transplanted mouse and human islets when grafted into mice. These findings suggest a novel strategy with potentially great impact to improve the efficiency of islet transplantation in clinical settings by targeting donor islets rather than recipients.


Abbreviations
IDDM

insulin-dependent diabetes mellitus

HMGB1

high-mobility group box 1 protein

NCX

Na+/Ca2+ exchanger

STZ

streptozotocin.

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. References
  10. Supporting Information

Pancreatic islet transplantation has now become a procedure of choice for the treatment of insulin-dependent diabetes mellitus (IDDM) since Shapiro et al. [1] reported that insulin independence could be achieved after sequential transplantations of islets combined with a novel immunosuppressive regime. Since then, the rate of insulin independence among IDDM recipients has improved to reach 50–60% at 5 years after islet transplantation [2-4]. Currently, however, insulin independence has been achieved in limited patients with IDDM after islet transplantation from a single donor, and therefore sequential transplantation of islets with the use of 2–3 donors is still required for the treatment of a single recipient [1-4]. Thus, this low efficiency has been a major obstacle facing clinical islet transplantation.

Previously, we have shown in mice that the low efficiency of islet transplantation is mainly due to early graft loss in the liver, the site of islet transplantation. This is caused by innate immune rejection of transplanted islets in concert with activation of DC, NKT cells and neutrophils to produce IFN-γ [5], and is triggered by high-mobility group box 1 protein (HMGB1) released from islets soon after their transplantation [6]. Importantly, by targeting this HMGB1-DC-NKT cell-neutrophil-IFN-γ-mediated pathway, the efficiency of islet transplantation was sufficiently enhanced to the point that islet transplantation from one donor to one recipient became feasible in mice [5, 6]. However, the upstream events that cause HMGB1 release from transplanted islets soon after transplantation remain unknown.

Since the site of clinical islet transplantation is the portal vein in the liver [1-4], we speculated that transplanted islets are immediately exposed to low oxygen tension and therefore the transplanted β cells are easily damaged by hypoxia, which may induce HMGB1 release to activate innate immune cells thus causing the early loss of transplanted islets. Furthermore, accumulating evidence has shown that hypoxic cell death is caused by an increase in intracellular calcium ([Ca2+]i) [7], suggesting that pathways involved in the influx of calcium ions may be a therapeutic target of intervention. Among calcium channels and transporters, we focused on the sodium (Na+)-calcium (Ca2+) exchanger (NCX), since inhibition of this membrane protein has been reported to help prevent cell death resulting from ischemia-reperfusion injury [8-10].

NCX is a major Ca2+-regulatory protein expressed by all excitable and many nonexcitable cells and transports Ca2+ across the plasma membrane [11]. Pancreatic islet β cells express NCX [12] and it participates not only in the maintenance of glucose homeostasis by regulated Ca2+-dependent secretion of insulin [13], but also in cell survival or death through its anti- or pro-apoptotic effects, respectively [14, 15].

In the present study, we hypothesized that NCX on transplanted islets mediates ischemic islet cell death soon after transplantation, and that this outcome might be prevented by in vitro pretreatment of donor islets prior to transplantation with a specific NCX inhibitor. This is indeed the case; thus, the present study identifies a novel strategy to improve the efficiency of islet transplantation by targeting donor islets rather than recipients.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. References
  10. Supporting Information

Mice

Male C57BL/6 and NOD/scid mice were purchased from Charles River Japan (Kanagawa, Japan). Mouse insulin promoter (MIP)-GFP mice (C57BL/6 background) [16] were purchased from Jackson Laboratory. Mice were kept under specific pathogen-free conditions and used at 8–16 weeks of age. All experiments were in accordance with protocols approved by the Animal Care and Use Committee at Fukuoka University.

Mouse islet isolation and in vitro culture

Islets were isolated from the mouse pancreas [17, 18] and those with a diameter of 150–250 μm were hand-picked and used for the experiments. Isolated islets were cultured at 24°C in a CO2-incubator (5% CO2 + 95% air) in medium (D-MEM, Nissui) supplemented with 10% FBS. After overnight culture, the medium was changed to that containing 0.2% bovine serum albumin (BSA, Sigma, St. Louis, MO) and islets were further incubated for 3 h in medium containing a specific NCX inhibitor, SEA0400 [8] or vehicle (DMSO). Islets were then cultured for 6–12 h at 37°C in the normoxic CO2-incubator (95% air, 5% CO2) or in a hypoxic chamber (Belco, Vineland, NJ) gassed with 1% O2 + 5% CO2 + 94% N2.

Fluorescent micrographs

Islets were stained with HO342 (Sigma) and PI (Sigma) and observed under fluorescent microscopy.

HMGB1 measurement

The amount of HMGB1 in the culture medium and the serum of recipient mice was measured with an ELISA kit (Shino-test Co., Sagamihara, Japan) [19].

Ca imaging

Isolated islets from the MIP-GFP mouse pancreas were further dispersed into single cells [20]. The cells were incubated in Krebs Ringer bicarbonate buffer (HKRB) with 1 μM Fura-2AM (Invitrogen, Carlsbad, CA) for 30 min at 37°C. To measure the [Na+]i-dependent Ca2+ influx, cells were perfused with Ca2+-free HKRB for 20 min and then switched to a Na+-free buffer HKRB in the presence of 1 μM ouabain. Fura-2AM was excited at 340 and 380 nm, and the emissions at >510 nm were captured every 500 ms using an Aquacosmos system (Hamamatsu Photonics, Hamamatsu, Japan).

Mouse islet transplantation

The 200 C57BL/6 islets pretreated with SEA0400 or DMSO alone were transplanted into the liver via the portal vein [21] of syngenic recipient mice made diabetic with STZ (Sigma) (180 mg/kg). The nonfasting plasma glucose levels were measured three times a week after the injection of STZ and once a week after islet transplantation using a GlucoCard DIA meter (Arkray, Kyoto, Japan).

Liver MNC preparation and flow cytometry

Preparation of liver MNCs and antibodies used are described in the Supplemental Materials and Methods.

Human islet experiments

Human islets were provided by Prodo Lab (Irvine, CA) and were cultured at 24°C in CMRL1066 medium (Mediatech, Manassas, VA) containing 0.2% human albumin for 2–3 days prior to the following experiments.

For islet transplantation, three batches of human islets in total were used (Table S1). For each batch, one or two transplants using SEA0400- or vehicle-treated islets (1000 IEQ) were performed. Human islets were cultured in the presence of 10 μM SEA0400 or vehicle (DMSO) for 24 h and grafted beneath the capsule of one kidney of STZ (170 mg/kg, iv injection)-induced diabetic male NOD/scid mice [22]. Nonfasting plasma glucose and body weight were measured once a week after transplantation. The kidneys of recipient mice bearing human islets were removed and fixed in 10% formalin, embedded in paraffin and sectioned for histological analysis.

Statistical analysis

The statistical significance of differences was determined by a one- or two- tailed Student's t-test. The statistical significance with respect to the rate of normoglycemia in streptozotocin-induced diabetic NOD/scid mice after transplantation of human islets was determined by Fisher's exact test.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. References
  10. Supporting Information

HMGB1 release from hypoxia-damaged islets is prevented by SEA0400, a specific NCX inhibitor

In order to investigate whether the early loss of islets after transplantation is caused by hypoxia-mediated damage to the islet cells during transplantation, we first focused on the Na+/Ca2+ exchanger (NCX), which plays a central role in elevating intracellular calcium during ischemia and reperfusion in various tissues [8-10].

Three isoforms of NCX have been described [11]; therefore, our initial experiment was to determine which NCX type is expressed on and functions in mouse pancreatic islet cells. A quantitative real-time PCR analysis revealed that islet cells predominantly express Ncx1 transcripts (Figure S1a), which is reflected in the predominance of NCX1 protein expression in β cells (Figure S1b). To address the question of whether a specific NCX inhibitor, SEA0400 [8], can suppress NCX function in β cells, single cells from mouse islets genetically engineered to express GFP under the MIP [16] were prepared and assayed (Figure 1a). The intracellular Ca2+ concentration ([Ca2+]i) of GFP+ β cells was stable when the cells were cultured in Na+-containing medium, but increased in Na+-free medium (Figure 1b). This increase in [Ca2+]i was almost completely blocked by the addition of SEA0400 (Figure 1b). The Ca2+ influx via NCX on islet cells was also confirmed under the conditions of hypoxia/reoxygenation by measuring Ca2+ influx in the MIN6 islet cell line [23] using the calcium indicator GCaMP2 [24] (Figure S2a).

image

Figure 1. The function of NCX is blocked by the specific NCX inhibitor, SEA0400. (A) Fluorescent-micrographs of an isolated islet (left) and dispersed islet single cells (right) from MIP-GFP mice. Original magnification; x200. Bars indicate 50 (left) and 10 μm (right). (B) [Na+]i-dependent Ca2+ influx via NCX in GFP+-β cells. Fura-2AM-loaded cells were exposed to Ca2+-free HKRB for 20 min and then to Na+-free HKRB, with or without 10 μM SEA0400. Typical traces (left panel) and average data (right panel, n = 3) of Ca2+ signals were shown in the left and right column, respectively. Data are expressed as the mean ± SE of Ca2+ influx at 1 and 3 min. *p < 0.01 (n = 3) and **p < 0.05 (n = 3).

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Since NCX on β cells is functional and its function can be blocked by SEA0400, we investigated whether pretreatment of islets with SEA0400 prior to hypoxic culture has any beneficial effect on their survival. For these studies, isolated islets were cultured at 37°C inside a chamber filled with hypoxic gas (1%O2, 5%CO2 and 94%N2). Under these conditions, the O2 concentration in the culture medium inside the chamber dropped to 20 mmHg within 30 min and was maintained at that level for more than 12 hrs (Figure S2b). As a control for normoxia conditions, islets were cultured at 37°C in a CO2-incubator gassed with 5% CO2 and 95% air. Islet cell death was assessed by fluorescent microscopy using the DNA binding dyes propidium iodide (PI) and Hoechst 33342 (HO 342) [25]; the nuclei of all cells are stained blue by HO342, whereas only dead cell nuclei are stained red by PI. When isolated islets were cultured under normoxia conditions, only a few PI positive cells were observed (Figure 2a, left column). In marked contrast, under hypoxic conditions the number of PI positive islet cells was significantly increased at 6 h (Figure 2a, right upper panel). When isolated islets were incubated under normoxia conditions for 3 h in the presence of SEA0400 (10 μM), then transferred into the hypoxic chamber and cultured thereafter in the absence of SEA0400, the number of PI positive cells was markedly decreased at 6 h compared with islets treated with vehicle, dimethyl sulfoxide (DMSO) (Figure 2a, right, middle, and lower panels).

image

Figure 2. Treatment with the NCX-specific inhibitor, SEA0400, helps prevent in vitro hypoxic islet cell death. (A) Fluorescent micrographs of islets without treatment (upper panel) and pretreated with DMSO (middle panel) or SEA0400 10 μM (lower panel), cultured in normoxia (left columns) or in hypoxia (right columns) for 6 h and stained with HO342 (blue) and PI (red). A representative micrograph of an islet in each group is shown. The experiment was repeated three times. Original magnification; 200×. Bars indicate 50 μm. (B) The amount of HMGB1 in the culture medium of islets in normoxia versus hypoxia measured by ELISA. Islets pretreated with 10 μM SEA0400 or vehicle (DMSO) were cultured in hypoxia (open triangle and square) or normoxia (open circle) and the medium was collected at the times indicated. The values are expressed as the mean ± SD (n = 5). *p < 0.01. **p < 0.001. Representative data of the two experiments are shown.

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In order to validate the beneficial effect of SEA0400 on islet cell survival, we measured the amount of HMGB1 in the culture medium, since our previous study demonstrated that HMGB1 is abundant within the islets and is released into the medium in parallel with the degree of β cell damage [6]. The amount of HMGB1 in the medium of islets pretreated with SEA0400 followed by culture under hypoxia conditions was significantly lower than that seen when islets were similarly cultured but pretreated with DMSO (Figure 2b), clearly demonstrating that the pretreatment of islet cells with the NCX inhibitor maintained viability and inhibited HMGB1 release.

Pretreatment of donor islets with SEA0400 in vitro prevents their early loss after transplantation

The above results strongly suggested that pretreatment of islets with SEA0400 in vitro might prevent their early loss after transplantation in vivo. To assess possible beneficial effects of SEA0400-pretreatment, streptozotocin (STZ)-induced diabetic mice were transplanted with 200 islets from a single donor, a dose that is normally not sufficient to normalize hyperglycemia [5, 6]. As expected, the diabetic mice receiving 200 islets pretreated with DMSO remained hyperglycemic (Figure 3a, upper panel). In striking contrast, diabetic recipients of 200 islets pretreated with 10 μM SEA0400 for 3 h prior to transplantation became normoglycemic (Figure 3a, lower panel). These findings suggest that islets pretreated with SEA0400 in vitro prior to transplantation are less damaged in vivo, resulting in the inhibition of HMGB1 release and protection from attack by the innate immune system.

image

Figure 3. Pretreatment of donor islets with SEA0400 in vitro prevents their early loss after transplantation. (A) Nonfasting plasma glucose levels of STZ-induced diabetic mice (C57BL/6) transplanted with 200 syngeneic islets pretreated with DMSO (upper panel) or 10 μM SEA0400 (lower panel). Individual lines represent glucose levels of each animal. (B) Serum concentration of HMGB1 in diabetic mice receiving 400 islets pretreated with SEA0400 or vehicle (DMSO) at 6 h after transplantation. In this particular experiment, 400 islets were used so that the islet-derived HMGB1 could be readily detected. The values are expressed as the mean ± SD in each group (n = 4). *p < 0.005. (C) FACS analysis of liver MNCs from naïve mice (first row) and from diabetic mice at 6 h after transplantation of 200 syngeneic islets pretreated with vehicle (DMSO) (second row) or with 10 μM SEA0400 (third row). NKT (CD1d-dimer+ CD3+) cells and Gr-1+ CD11b+ cells were analyzed for IFN-γ production (second and fourth columns). Representative data from two to three experiments are shown.

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In fact, when the SEA0400-pretreated islet cells were transplanted, the cellular cascade of HMGB1-mediated NKT cell-dependent IFN-γ production by Gr-1+ CD11b+cells (neutrophils), which is essential for the early loss of transplanted islets [5], was not activated. The serum level of HMGB1 at 6 h after transplantation of SEA0400-pretreated islets was significantly lower than those of control mice receiving DMSO-treated islets (Figure 3b). Moreover, there was no detectable IFN-γ production by NKT cells or neutrophils in mice receiving SEA0400-pretreated islets, whereas it was up-regulated in both cell types in mice receiving control islets (Figure 3c).

In order to confirm that NCX actually plays an essential role in the early loss of transplanted islets, we used genetically engineered Ncx1+/− GFP+ β cells obtained by crossing Ncx1+/− [26] and MIP-GFP mice [16] and found that the function of NCX is suppressed and that Ncx1+/− islets, similar to wild-type islets treated with SEA0400, are resistant to in vitro and in vivo hypoxic cell death (Figure S3).

One possibility for the amelioration of hyperglycemia in diabetic mice receiving donor islets pretreated with SEA0400 was augmented insulin release from transplanted islets. Therefore, we examined whether the SEA0400 treatment alters the amount of insulin released from islets in vitro in response to glucose and found that there was no effect (Figure S4), indicating that the beneficial effect of SEA0400 is due to prevention of early graft loss.

Beneficial effect of SEA0400 pretreatment on human islets grafted into NOD/scid mice

A key question that we next investigated was whether the remarkable effect of SEA0400 pretreatment on survival of mouse islets would translate to human islets. Prior to transplantation experiments, we confirmed the expression of NCX1 by human islets and found that, as in mice, human islet cells predominantly express NCX1 transcripts (Figure 4a) and protein (Figure 4b). In the islet transplantation experiments, human (Figure 4c) or mouse (Figure S5) islets were grafted beneath the kidney capsule of streptozotocin-induced diabetic NOD/scid mice. When the mice received 1000 IEQ (islet equivalent) human islets pretreated with SEA0400 prior to transplantation, their plasma glucose levels (5/5) became less than 250 mg/dL by 30 days after transplantation (Figure 4c), while the diabetic mice receiving vehicle (DMSO) treated islets (4/5) remained hyperglycemia. The difference in the rate of normoglycemia between the two groups of recipient mice was statistically significant (p < 0.05, by Fisher's exact test). In order to confirm that the normoglycemia of recipient mice is dependent on transplanted human islets, the kidneys bearing human islet grafts were removed at 70–100 days after transplantation, which promptly made the mice hyperglycemic again (Figure 4c, left panel). Morphologically, insulin-containing cells were abundant beneath the kidney capsule of normoglycemic (Figure 4c, right panel) compared with hyperglycemic recipient mice.

image

Figure 4. Beneficial effect of SEA0400 on engraftment of human islets in NOD/scid mice. (A) A quantitative real-time PCR analysis revealed that human islet cells predominantly express NCX1 transcripts. (B) Immunohistochemical study of human pancreata revealed that human islet β cells express NCX1 protein. (C) Isolated human islets and streptozotocin-induced diabetic NOD/scid mice were used as donors and recipients, respectively. Individual lines represent the nonfasting plasma glucose levels of diabetic NOD/scid mice transplanted with 1000 IEQ (islet equivalent) human islets pretreated with SEA0400 or vehicle (DMSO) prior to transplantation (left column). *The kidneys bearing human islet grafts were removed on the day indicated. Human islet grafts pretreated with SEA0400 were retrieved at 72 days after transplantation, fixed and immunolabeled with anti-insulin (green) and anti-glucagon (red) antibodies, counterstained with DAPI (blue) (right column). Scale bars represent 50 μm. The boxed area in the left image is shown at a higher magnification on the right.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. References
  10. Supporting Information

The findings presented here demonstrate that the release of HMGB1, an initial trigger of early loss of transplanted islets, is due to hypoxic death of the islet cells. This is mediated by the increase in intracellular Ca2+ concentration of β cells through NCX1 and can be prevented by a specific NCX inhibitor, SEA0400. Importantly, the hyperglycemia of STZ-induced diabetes was ameliorated by transplantation of 200 mouse islets pretreated with SEA0400 in vitro, whereas the mice remained hyperglycemic if transplanted with 200 vehicle-treated islets. Furthermore, we found that human islet cells also predominantly express NCX1 and that the loss of human islet grafts is also prevented by pretreatment of the donor islets with SEA0400 in vitro prior to transplantation into immunodeficient mice. These preclinical results are quite promising and strongly suggest that the use of a NCX inhibitor could be applied to human donor islets to improve efficiency of islet transplantation in a clinical setting.

The early loss of transplanted islets has been a major concern to improve the efficiency of islet transplantation. Although our current study unveils the upstream event as to etiology in association with HMGB1 release from transplanted islets triggering innate immune responses in the liver, the site of islet transplantation, it is a matter of interest for the future study to dissect the roles of other inflammatory cytokines and chemokines in the early loss of transplanted islets in relation to HMGB1-mediated pathway. Furthermore, it is interesting to investigate whether the treatment with the NCX inhibitor improves the viability of islets and/or rescues their damage under stress conditions with a more comprehensive method as described [27].

Thus, the present study indicates that if the beneficial effect of the specific NCX inhibitor SEA0400 on the outcome of islet transplantation is also indeed the case clinically, it will have a tremendous impact on the treatment of patients with IDDM.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. References
  10. Supporting Information

We are grateful to Peter Burrows for helpful comments in the preparation of the manuscript. We thank I Komuro for providing Ncx1+/− mouse, J Miyazaki for the MIN6 β cell line, K Ishii for the support of Ca2+ measurements. We also thank T Ogata and M Nagaishi for their technical assistance. This work was supported by Global FU Program from Fukuoka University (Y.Y.), a Grant in Aids for Scientific Research from Japan Society of the Promotion of Science (Y.Y.), and for Scientific Research on Innovative Area from Ministry of Education, Culture, Sports, Science and Technology, Japan (S. Kita), Health Science Research Grant from the Ministry of Health, Labor and Welfare, Japan (Y.Y.), a grant from the Vehicle Racing Commemorative Foundation (T. Iwamoto) and Shimura Memorial Foundation (Y.Y.).

Disclosures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. References
  10. Supporting Information

The authors of this manuscript have no conflict of interest to disclose as described by the American Journal of Transplantation.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. References
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. References
  10. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
ajt12306-sm-0001-SuppData.docx1407K

Figure S1: Ncx1 is the predominant Ncx isoform expressed by mouse islet cells. (A) RT-PCR analysis of Ncx1, Ncx2 and Ncx3 expression. Isolated mouse islets (1000) were analyzed for mRNA levels by quantitative real-time PCR. Brain tissues and Gapdh were used as controls. (B) Fluorescent-micrographs of islets. Islets from wild-type C57BL/6 mouse pancreas were examined. The frozen sections were stained with DAPI (left column), anti-insulin (second column) and anti-NCXs (third column) (Suppl ref 1). The merged images are shown in the fourth column. Original magnification: 200×. Bars represent 50 μm.

Figure S2: NCX on MIN6 insulinoma cells functions during hypoxia/reoxygenation and is suppressed by a specific inhibitor, SEA0400. (A) [Ca2+]i elevation induced by hypoxia/reoxygenation in MIN6 cells is suppressed by a specific NCX inhibitor SEA0400. Typical traces (left panel) and average data (right panel) of Ca2+ signals when MIN6 cells expressing the Ca-indicator GCaMP2 were exposed to 5% CO2/95% air after culturing under hypoxic conditions for 6 h. The cells were treated with SEA0400 (10 μM) or vehicle. Each column and bar represent the mean ± SD of individual cells (n = 6–7). The difference between the two groups was statistically significant (*p < 0.001). (B) Oxygen tension in the medium during culture in hypoxia. Culture dishes were placed inside the chamber maintained at 37°C and gassed with 1% O2, 5% CO2 and 94% N2 and oxygen tension of the medium was measured continuously for more than 12 h using polarography (Unique Medical).

Figure S3: Use of Ncx1+/− islets to examine the role of NCX1 in in vitro hypoxic death and in vivo islet cell death after transplantation. (A) Dispersed cultured single cells prepared from Ncx1+/+ or Ncx1+/− MIP-GFP+ islets were used for the experiments. Typical traces (left panel) and average data (right panel) of Ca2+ signals when Ncx1+/− MIP-GFP+ islets were exposed to 5% CO2/95% air after hypoxic conditions for 6 h are shown. Each column and bar represents the mean ± SE at the time indicated. The differences were statistically significant compared with the corresponding controls (*p < 0.01, n = 3 and **p < 0.05, n = 3). (B) Fluorescent-micrographs of WT and Ncx1+/− islets cultured for 6 h in normoxia or in hypoxia as indicated. Cultured islets were stained with HO342 and PI. Representative micrographs of WT and Ncx1+/− islets are shown. Original magnification, 200×. Bars indicate 100 μm. (C) Nonfasting plasma glucose levels of STZ-induced diabetic mice transplanted with 200 syngeneic WT or Ncx1+/− islets. Lines represent individual animals. (D) Serum HMGB1 concentrations of mice receiving WT or Ncx1+/− islets at 6 h after transplantation. The difference was statistically significant (*p = <0.01, n = 3 in each group). (E) Flow cytometry profiles of MNC from the livers of mice receiving 200 WT or Ncx1+/− islets at 6 h after transplantation into the liver. Representative data of two experiments are shown.

Figure S4: The amount of insulin released from wild-type islets in vitro in response to glucose was not affected by treatment with SEA0400. Isolated islets were cultured overnight and used for the in vitro experiments. Islets were maintained at 37°C in a CO2-incubator (95% air + 5% CO2) throughout the experiment. Islets were first preincubated with KRH buffer containing 50 mg/dL glucose supplemental with 0.2% BSA for 2 h. Then, islets were further incubated in the KRH buffer containing 50 (n = 4) or 300 mg/dL (n = 4) glucose for 1 hour. The amount of insulin released in the medium after the last incubation was measured by ELISA (Morinaga, Tokyo, Japan). The values are expressed as mean ± SD (n = 4).

Figure S5: In vitro pretreatment of donor islets with SEA0400 helps prevent early loss of islet grafts transplanted beneath the kidney capsule of diabetic recipient mice. Nonfasting plasma glucose levels of STZ-induced diabetic mice transplanted with 50 syngeneic WT islets treated with 10 μM SEA0400 or vehicle (DMSO) prior to transplantation are shown. Lines represent plasma glucose levels of individual mice. *The kidneys bearing islet grafts were removed at the day indicated.

Table S1: Donors and islet characteristics

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