Clinical islet transplantation has become an established treatment modality for selected patients with type 1 diabetes. However, a large proportion of transplanted islets is lost through multiple factors, including immunosuppressant-related toxicity, often requiring more than one donor to achieve insulin independence. On the basis of the cytoprotective capabilities of antifreeze proteins (AFPs), we hypothesized that supplementation of islets with synthetic AFP analog antiaging glycopeptide (AAGP) would enhance posttransplant engraftment and function and protect against tacrolimus (Tac) toxicity. In vitro and in vivo islet Tac exposure elicited significant but reversible reduction in insulin secretion in both mouse and human islets. Supplementation with AAGP resulted in improvement of islet survival (Tac+ vs. Tac+AAGP, 31.5% vs. 67.6%, P < 0.01) coupled with better insulin secretion (area under the curve: Tac+ vs. Tac+AAGP, 7.3 vs. 129.2 mmol/L/60 min, P < 0.001). The addition of AAGP reduced oxidative stress, enhanced insulin exocytosis, improved apoptosis, and improved engraftment in mice by decreasing expression of interleukin (IL)-1β, IL-6, keratinocyte chemokine, and tumor necrosis factor-α. Finally, transplant efficacy was superior in the Tac+AAGP group and was similar to islets not exposed to Tac, despite receiving continuous treatment for a limited time. Thus, supplementation with AAGP during culture improves islet potency and attenuates long-term Tac-induced graft dysfunction.

Islet transplantation outcomes have improved significantly in highly specialized centers, with a 5-year insulin independence rate exceeding 50% (1). However, a proportion of patients require reintroduction of insulin at delayed time points (2). Numerous factors contribute to limited durability of both short- and long-term glycemic control. Exposure to diabetogenic immunosuppressive agents is associated with islet functional impairment and graft loss, especially that linked to corticosteroid exposure or use of calcineurin inhibitors (CNIs), including tacrolimus (Tac) (3,4). Tac is the most potent mainstay immunosuppressant in most clinical protocols, although diabetogenicity is well documented, accounting for an increased incidence of posttransplant diabetes mellitus (58). Early peak Tac levels in portal blood after islet transplantation after oral administration may further increase islet exposure, thereby magnifying toxicity (9,10). Although undesirable, Tac has become necessary to preventing rejection and autoimmune recurrence after islet transplantation and may be partly responsible for limited islet durability and a need for multiple donors for each recipient (11).

Antifreeze proteins (AFPs) have generated considerable interest for their ability to protect cells under a variety of conditions. They naturally occur in Arctic and Antarctic fish as well as in other cold climate–dwelling invertebrates and are responsible for maintaining cell and tissue function at subzero temperatures (12,13). AFPs were successfully isolated in the 1950s and have demonstrated an ability to noncolligatively lower the freezing temperature of body fluids by binding to ice crystals (12,14). Early experiments with AFPs in the field of organ and tissue transplantation showed promising results, making them attractive therapeutic candidates to protect cells against harmful conditions associated with procurement, preservation, and reperfusion (14). Moreover, benefits have been demonstrated during cryopreservation of various cells, including islets of Langerhans, with significant improvements in their viability and function when supplemented with AFP during cryostasis (15,16).

Antiaging glycopeptide (AAGP) is a 580-Da synthetic AFP analog initially developed by Géraldine Castelot-Deliencourt-Godefroy (Rouen, France) and later manufactured by ProtoKinetix Inc. (Vancouver, BC, Canada). This new compound has improved stability, is water soluble, and has proven to be more potent in terms of cytoprotective capabilities under extreme conditions (pH variations, sudden temperature changes, nutrient deprivation, oxidative stress, ultraviolet radiation, and inflammation) (17).

In light of this evidence, significant attention is now being directed toward AFPs and their potential use in reparative and regenerative medicine, particularly in the field of transplantation. We evaluated the cytoprotective capacity of AAGP to protect against the diabetogenic effect of Tac, resulting in improved islet engraftment.

Human Islet Isolation, Purification, and Culture

Human islet preparations were isolated from consenting deceased multiorgan donors, as previously described (18), with intent for clinical transplantation and were only made available for research when the islet yield fell below that of the minimal mass required. Permission for these studies was granted by the Health Research Ethics Board of the University of Alberta, Edmonton, AB, Canada, and after written permission from donor families.

In Vitro Evaluation of AAGP

Islet Recovery, Viability, and Secretory Function

Experiments with human islets in vitro included four groups: 1) control (islets cultured in medium alone) (Tac), 2) islets cultured in medium containing AAGP (AAGP), 3) islets cultured in medium containing Tac (Prograf; Astellas Pharma Canada Inc., Markham, ON, Canada) (Tac+), and 4) islets cultured in medium supplemented with AAGP (ProtoKinetix) and Tac (Tac+AAGP). Islets were cultured for 24 h with ±3 mg/mL AAGP before addition of Tac at a clinically relevant concentration of 10 ng/mL. All four groups were then cultured for an additional 24 h.

Islets were assessed for recovery, viability, insulin release, oxidative stress, and cell death. Recovery rate was calculated as the percentage of surviving islets after culture compared with the initial count for each condition. Viability was assessed by fluorescent membrane integrity assay with counterstains using SYTO 13 green fluorescent nucleic acid stain (Life Technologies, Burlington, ON, Canada) and ethidium bromide (Sigma-Aldrich, ON, Canada) (1922).

Hormonal islet secretory function was assessed by both static glucose-stimulated insulin secretion (s-GSIS), sequentially performed at low (2.8 mmol/L) and high (16.7 mmol/L) glucose concentrations and by dynamic islet perifusion (D-IP), as described by Cabrera et al. (23). D-IP was performed at 16-min intervals by using low (2.8 mmol/L) then high × 2 (28 mmol/L) followed by low glucose concentrations. Glucose was infused at 100 μL/min, and results are expressed as fold change of insulin secretion compared with the low-glucose–stimulation baseline normalized for 100 islet equivalents (IEQ). For s-GSIS, insulin concentrations in supernatants were measured by ELISA (Mercodia, Uppsala, Sweden). A stimulation index was calculated as the ratio of stimulated to basal insulin secretion normalized by DNA. These insulin secretion studies were always performed in vitro on cultured human islets.

Apoptosis was assayed by determining the quantity of cleaved caspase-3 in the frozen lysates from fixed islets cultured under the aforementioned conditions by using a spectrophotometric assay (EMD Millipore, Billerica, MA). Results are expressed as fold-change increase compared with control.

Islet apoptosis was also examined by TUNEL staining (DeadEnd Colorimetric Apoptosis Detection System; Promega, Madison, WI) after formalin fixation, processing, and paraffin embedding. Costaining with insulin (1:200 concentration of anti-insulin antibody [Dako, Mississauga, ON, Canada] and DAPI [Invitrogen Molecular Probes, Eugene, OR]) was performed to identify grafts and nuclei, respectively. ImageJ version 1.33 software with Cell Counter plug-in (http://rsb.info.nih.gov/ij) was used to quantify islet apoptosis by percentage of positive TUNEL-stained area.

Reactive Oxygen Species Analysis

Frozen samples from the study groups were assayed for reactive oxygen species (ROS) released into the culture media by Acridan Lumigen PS-3 assay (Amersham ECL Plus Kit; Fisher Scientific Inc., Ottawa, ON, Canada) (24). Acridan Lumigen PS-3 is excited by ROS and reactive nitrogen species in the presence of hydrogen peroxide, producing chemiluminescense at 430 nm. Media samples were flash frozen in liquid nitrogen and stored until analyzed. Connaught Medical Research Laboratories culture medium alone served as control, and results were expressed as fold-change increase compared with control.

Mixed Lymphocyte Reaction

To rule out direct drug inhibition of AAGP and Tac, a one-way mixed lymphocyte reaction (MLR) assay was performed to assess proliferative response of responder T cells against antigens present on allogeneic stimulator cells. T cells were isolated from γ-irradiated splenocytes of BALB/c mice (stimulators) and C57BL/6 mice (responders). Proliferation was measured by loss of fluorescence intensity by using fluorochrome 5,6-carboxyfluorescein diacetate succinimidyl ester (Invitrogen), which spontaneously binds to intracellular proteins shared between daughter cells. Controls (Tac, AAGP), Tac+, and Tac+AAGP groups were studied. T-cell proliferation was measured by flow cytometry after characterizing different subpopulations by cell surface antibody staining with anti-mouse T-cell receptor-β eFluor450, CD4 antigen-presenting cell, and CD8a antigen-presenting cell eFluor 780 (eBioscience, San Diego, CA). Acquisition was performed on a BD LSR II flow cytometer (BD Biosciences, Franklin Lakes, NJ), followed by analysis with FCS Express flow cytometry software (De Novo Software, Los Angeles, CA).

Calcium Imaging

Measurements of intracellular calcium concentration for individual human islets from the different treatment groups were carried out by previously described methods (25,26) at glucose concentrations of 2.5 and 25 mmol/L. Glucose-stimulated increase in intracellular calcium concentration was expressed as area under the curve (AUC).

Capacitance Studies

Measurement of membrane capacitance was performed on islets according to our previously established method (27,28) to determine the effect of Tac and AAGP on β-cell exocytotic responses. Cells were stimulated with a series of 10 depolarizations to activate voltage-dependent Ca2+ channels. Whole-cell capacitance responses were normalized to initial cell size and expressed as femtofarad per picofarad (fF/pF).

In Vivo Evaluation

All mice were housed under conventional conditions, with access to food and water ad libitum. The care for mice was in accordance with guidelines approved by the Canadian Council on Animal Care.

Transplants With Human Islets and Inflammation Analysis

Diabetes was induced chemically in 8- to 12-week-old immunodeficient B6.129S7-Rag1tm1Mom recipient mice (The Jackson Laboratory, Bar Harbor, ME) by intraperitoneal injection of 180 mg/kg streptozotocin (Sigma-Aldrich). Mice were considered diabetic after two consecutive blood glucose measurements ≥11.3 mmol/L (350 mg/dL). Recipients (n = 10 per group) received ∼1,000 IEQ human islets from three different isolations. Islets from each isolation were randomly allocated to each group (Tac, AAGP, Tac+, Tac+AAGP) to control for potential differences in each islet preparation. Islets were transplanted under the kidney capsule as described previously (29). A minimal islet dose was used to stress the model and maximize covert toxicity (3032).

Three mice per group underwent acute graft explantation and were killed on day 1 and 7 posttransplant to determine proinflammatory cytokine concentrations (at both time points), cleaved caspase-3, and TUNEL-stained area (24 h) within the graft. For cytokine and cleaved caspase-3 quantification, the islet grafts were excised from the kidney and bisectioned, with one section flash frozen in liquid nitrogen and stored at −80°C and the other formalin fixed and processed for TUNEL quantification. Tissue samples were subsequently lysed in acid buffer as reported previously (30). Cytokine and cleaved caspase-3 determination was adjusted per gram of tissue.

Proinflammatory Cytokines and Chemokines

Relevant cytokines and chemokines (interferon-γ, interleukin [IL]-1β, IL-6, IL-10, IL-12, keratinocyte-derived chemokine [KC], and tumor necrosis factor-α [TNF-α]) were measured with a multispot Mouse ProInflammatory 7-Plex Ultra-Sensitive Kit and analyzed on a SECTOR Imager (Meso Scale Discovery, Gaithersburg, MD). Results are expressed as absolute values (pg/mL), and normal renal tissue lysate samples from a mouse receiving a sham operation were used as control.

Apoptosis

Apoptosis was determined in the excised grafts 24 h posttransplant by quantifying cleaved caspase-3 and analyzing percentage of death cells (TUNEL) within the graft. Caspase-3 concentration was expressed as fold-change increase compared with normal renal tissue lysate samples from a naive mouse.

Long-term Human Islet Graft Function After Transplantation in Immunodeficient Mice

Nonfasting blood glucose was monitored in the remaining animals three times a week with a portable glucometer (OneTouch Ultra2; LifeScan Canada) over 60 days. Normoglycemia was defined as two consecutive readings <11.3 mmol/L.

Intraperitoneal glucose tolerance tests (IPGTTs) were conducted 60 days posttransplant to evaluate the capacity of islets to respond to a glucose bolus (3 g/kg) after overnight fast. Blood glucose levels were monitored at baseline time 0 and 15, 30, 60, 90, and 120 min postinjection. All results were compared with blood glucose profiles of naive control nondiabetic mice.

Recovery islet–bearing nephrectomies were performed on day 65 to demonstrate graft-dependent euglycemia. Both cultured islets and recovered grafts were stored at −80°C and processed to measure intracellular insulin content by acid-ethanol homogenization and ultrasonic lysis. The extract was neutralized and insulin measured with ELISA (30).

Transplants With Mouse Islets Under Continuous Treatment With Tac

Mouse Islet Isolation

Pancreatic islets were isolated from 8- to 12-week-old male BALB/c mice (The Jackson Laboratory), as reported previously (18). Islets were counted and divided into three groups (Tac, Tac+, and Tac+AAGP). All islets were incubated for 1 h under the previously described conditions, and Tac+AAGP islets were supplemented with AAGP at 3 mg/mL during the incubation period. Recipient syngeneic BALB/c mice were also rendered diabetic with streptozotocin and transplanted after incubation with 500 IEQ ± 10% of 90% purity under the renal capsule (18).

Subcutaneous micro-osmotic pumps (model 1002; ALZET, Cupertino, CA) were implanted in all mice at the time of islet transplantation to provide continuous delivery of Tac. A first group (short duration) received a pump delivering Tac for 7 days at a dose of 1 mg/kg/day (Tac+ [n = 6], Tac+AAGP [n = 8]), and a second group (long duration) received pumps delivering the CNI for 28 days (Tac+ [n = 6], Tac+AAGP [n = 6]). The Tac group (n = 10) received pumps loaded with normal saline as placebo. Steady-state Tac levels were monitored selectively from the dorsal tail vein at day 5 and ranged from 10 to 20 ng/mL (clinically relevant range, tandem liquid chromatography–mass spectrometry for continuously administered drug) (33).

Animals underwent IPGTT on day 7 (short duration) and day 14 (long duration) during the treatment course and again on days 30 and 40 after CNI treatment cessation. Transplant islet–bearing nephrectomies were performed after tolerance tests to prove graft-dependent function.

Statistical Analysis

Data are presented as mean ± SEM. AUC was calculated for GSIS and D-IP, calcium imaging, capacitance measurements, and IPGTT, and differences between groups were analyzed with one-way ANOVA with Tukey post hoc test. P < 0.05 was considered significant, and all analyses were performed with GraphPad Prism software (GraphPad Software, La Jolla, CA).

AAGP Enhances Human Islet Potency in Culture and Protects Islets Against Acute Exposure to Tac

Isolated human islets from six different preparations were cultured in media supplemented with or without AAGP and Tac as described. After 48 h of culture, all groups were characterized for in vitro survival, viability, function, oxidative stress, and apoptosis.

After the study period, cells were counted, resulting in a greater number of surviving islets in the AAGP-supplemented group (71.1%). Exposure to Tac clearly decreased survival, but islets significantly recovered when simultaneously supplemented with AAGP (Tac+ vs. Tac+AAGP, 31.5% vs. 67.6%, P < 0.01) (Fig. 1A). There was no difference in cell viability by membrane integrity stain (Fig. 1B).

Figure 1

AAGP improves human islet potency in culture and protects against acute exposure to Tac. A: In vitro assessment of human islets in culture with or without AAGP supplementation showed a significantly higher islet recovery rate after culture in the presence of AAGP. B: No significant changes in cell viability were found after the study period. C and D: Perifusion curves comparing GSIS after stimulation with variable glucose concentrations (low 2.8 mmol/L, high 28 mmol/L) show severely impaired islet function for the Tac group and a significantly better response for groups treated with AAGP (also seen in the corresponding AUC, n = 6). Tac impairs insulin secretion without affecting insulin biosynthesis. s-GSIS assay and intracellular insulin content were simultaneously measured in human islets kept in culture. E: The stimulation index for the Tac+ group was significantly decreased compared with control. However, a significant improvement was observed in insulin secretion of Tac+AAGP islets (P < 0.01), whereas no changes were seen in the intracellular content of insulin across the different groups (F). Data are mean ± SEM (triplicates from four different preparations). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 1

AAGP improves human islet potency in culture and protects against acute exposure to Tac. A: In vitro assessment of human islets in culture with or without AAGP supplementation showed a significantly higher islet recovery rate after culture in the presence of AAGP. B: No significant changes in cell viability were found after the study period. C and D: Perifusion curves comparing GSIS after stimulation with variable glucose concentrations (low 2.8 mmol/L, high 28 mmol/L) show severely impaired islet function for the Tac group and a significantly better response for groups treated with AAGP (also seen in the corresponding AUC, n = 6). Tac impairs insulin secretion without affecting insulin biosynthesis. s-GSIS assay and intracellular insulin content were simultaneously measured in human islets kept in culture. E: The stimulation index for the Tac+ group was significantly decreased compared with control. However, a significant improvement was observed in insulin secretion of Tac+AAGP islets (P < 0.01), whereas no changes were seen in the intracellular content of insulin across the different groups (F). Data are mean ± SEM (triplicates from four different preparations). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

When comparing in vitro function by D-IP, insulin release was completely suppressed after Tac exposure (AUC: Tac vs. Tac+, 131 vs. 7.3 mmol/L/60 min, P < 0.001). However, islet function was fully restored after supplementation with AAGP and comparable to the other culture conditions (Tac+ vs. Tac+AAGP, 7.3 vs. 129.2 mmol/L/60 min, P < 0.001) (Fig. 1C and D).

Acute Exposure to Tac Decreases Insulin Secretion but Not Biosynthesis

Aliquots of 100 IEQ human islets were collected for each group for s-GSIS assay and intracellular insulin content. The Tac+ group showed significant impairment of insulin secretion, which was not observed in the Tac+AAGP group (stimulation index 1.4 vs. 10.7, P < 0.01) (Fig. 1E). However, intracellular insulin content remained stable and comparable across groups, demonstrating no changes to biosynthesis of insulin within β-cells (Fig. 1F).

AAGP Reduces Oxidative Stress but Does Not Inhibit Tac Function

Oxidative stress was observed in all groups, but Tac exposure resulted in a substantial increase in ROS, which was ameliorated in the presence of AAGP (n = 6, P < 0.05) (Fig. 2A). To confirm that AAGP did not inhibit Tac suppression of T-cell proliferation, MLRs were completed with donor splenocytes. The assay measured T-cell proliferative response by carboxyfluorescein diacetate succinimidyl ester staining. As expected, T-cell proliferation was significantly decreased in the presence of Tac compared with IgG controls (n = 4, P < 0.001). Proliferation of CD8+- and CD4+-positive T cells was also significantly decreased in the presence of Tac alone or in combination with AAGP (n = 4, P < 0.001 in both cases), with no impediment to MLR suppression in the presence of AAGP (Fig. 2B–D).

Figure 2

Islets treated with AAGP have decreased oxidative stress. A: Human islets in culture had an increased concentration of ROS when treated with Tac. However, supplementation with AAGP significantly decreased this effect (P < 0.05). Oxidative stress was measured by fold increase in extracellular ROS analyzed with the Acridan Lumigen PS-3 assay (n = 5). AAGP effect is not the result of direct drug inhibition with Tac. BD: Allogeneic MLR was used to evaluate direct drug inhibition. Results show a significant decrease of T-cell proliferation in the presence of Tac, AAGP, and the combination of both, hence no direct inhibition of Tac by AAGP. Data are mean ± SEM (n = 6). *P < 0.05, ***P < 0.001, ****P < 0.0001. TCR, T-cell receptor.

Figure 2

Islets treated with AAGP have decreased oxidative stress. A: Human islets in culture had an increased concentration of ROS when treated with Tac. However, supplementation with AAGP significantly decreased this effect (P < 0.05). Oxidative stress was measured by fold increase in extracellular ROS analyzed with the Acridan Lumigen PS-3 assay (n = 5). AAGP effect is not the result of direct drug inhibition with Tac. BD: Allogeneic MLR was used to evaluate direct drug inhibition. Results show a significant decrease of T-cell proliferation in the presence of Tac, AAGP, and the combination of both, hence no direct inhibition of Tac by AAGP. Data are mean ± SEM (n = 6). *P < 0.05, ***P < 0.001, ****P < 0.0001. TCR, T-cell receptor.

Tac Effect on Islet Intracellular Calcium Responses and Exocytosis

Various studies were performed on human islets to elucidate a potential mechanism of action for AAGP by characterizing CNI-related injury and its minimization. Intracellular calcium concentrations were measured to determine a possible influence of AAGP on glucose-stimulated calcium influx (34). No significant differences in calcium influx were observed between groups (AUC: Tac 209.4, Tac+ 221.6, Tac+AAGP 208.7 fF/treatment/2 s; P > 0.05), suggesting that the protective effect of AAGP was further downstream in the secretory pathway (Fig. 3A).

Figure 3

AAGP improves insulin release by increasing islet exocytosis. Human islets were cultured and treated with and without Tac/AAGP. A: Insulin secretion impairment for the Tac+ group was also met by a lower concentration of intracellular calcium. B: Differences among groups did not reach statistical significance, but further studies showed that normalized membrane capacitance measurements were significantly worse for Tac+ islets (A, blue), indicating impaired exocytosis, whereas measurements were superior and comparable in the Tac (A, red) and Tac+AAGP groups (A, black). Data are mean ± SEM (triplicates from two isolations). **P < 0.01, ***P < 0.001.

Figure 3

AAGP improves insulin release by increasing islet exocytosis. Human islets were cultured and treated with and without Tac/AAGP. A: Insulin secretion impairment for the Tac+ group was also met by a lower concentration of intracellular calcium. B: Differences among groups did not reach statistical significance, but further studies showed that normalized membrane capacitance measurements were significantly worse for Tac+ islets (A, blue), indicating impaired exocytosis, whereas measurements were superior and comparable in the Tac (A, red) and Tac+AAGP groups (A, black). Data are mean ± SEM (triplicates from two isolations). **P < 0.01, ***P < 0.001.

Complementary membrane capacitance studies were performed under similar conditions as an indirect indicator of insulin exocytosis. There was a decreased cumulative capacitance response in the Tac+ group compared with the other groups and a significantly lower AUC compared with the Tac+AAGP group (AUC 2.9 vs. 10.5 fF/pF/treatment, P < 0.001) (Fig. 3B).

AAGP Prevents Islet Apoptosis Resulting From In Vitro Exposure to Tac

Exposure to Tac during culture resulted in an increased concentration of intracellular cleaved caspase-3 (fold change: Tac vs. Tac+, 1.9 vs. 4.3, P < 0.05), which corresponded with increased the percentage of apoptotic islets (Tac vs. Tac+, 18.9% vs. 48.6%, P < 0.01). Conversely, pretreatment with AAGP prevented Tac-induced cell death, showing reduced levels of caspase-3 (Tac+ vs. Tac+AAGP, 4.3 vs. 2.2, P < 0.05) and a fewer number of apoptotic cells (Tac+ vs. Tac+AAGP, 48.6% vs. 26%, P < 0.05) (Fig. 4A and B). Representative slides from TUNEL histology are shown in Fig. 4C.

Figure 4

AAGP decreases islet loss in culture due to apoptosis, even in the presence of Tac. Human islets were cultured and treated with Tac/AAGP. Cell death due to apoptosis was significantly higher in Tac+ by quantification of intracellular cleaved caspase-3 (A) and by analysis of TUNEL staining (B). Supplementation of media with AAGP restored viability and significantly decrease cell death (P < 0.05). C: Representative TUNEL slides of fixed islets from different study groups. Arrows point to TUNEL-positive nuclei. *P < 0.05, **P < 0.01.

Figure 4

AAGP decreases islet loss in culture due to apoptosis, even in the presence of Tac. Human islets were cultured and treated with Tac/AAGP. Cell death due to apoptosis was significantly higher in Tac+ by quantification of intracellular cleaved caspase-3 (A) and by analysis of TUNEL staining (B). Supplementation of media with AAGP restored viability and significantly decrease cell death (P < 0.05). C: Representative TUNEL slides of fixed islets from different study groups. Arrows point to TUNEL-positive nuclei. *P < 0.05, **P < 0.01.

AAGP Ameliorates Inflammatory Response Immediately Posttransplant

Minimal-mass (1,000 IEQ) human islet transplants were performed in diabetic immunodeficient mice. Grafts from three animals per group (day 1 and day 7 posttransplant) were homogenized and characterized for proinflammatory cytokines and chemokines.

Acute levels of IL-1β were significantly increased in Tac+ with respect to those in the sham group (18.9 vs. 163.1 pg/g tissue, P < 0.001). Cytokine concentration, however, was considerably dampened in the Tac+AAGP group (163.1 vs. 44.9, P < 0.001), with similar excretion behavior on day 7 (269.5 vs. 121 pg/g tissue, n = 3, P < 0.001) (Fig. 5A and B). Similarly, IL-6 was significantly increased in the Tac+ group (1,414 vs. 804.7 pg/g tissue, n = 3, P < 0.001), but differences were no longer apparent at later time points (Fig. 5C and D).

Figure 5

AAGP ameliorates inflammatory response immediately posttransplant. Proinflammatory cytokines and chemokines locally expressed 24 h and 7 days after transplantation. Concentrations of IL-1β (A and B), IL-6 (C and D), KC/human growth-regulated oncogene (GRO) (E and F), and TNF-α (G and H) were significantly lower in the engrafted islets previously treated with AAGP. Cytokines were measured 24 h and 7 days after transplantation locally to the graft by homogenization (normalized per gram of tissue, n = 3). Data are mean ± SEM adjusted per gram of tissue (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 5

AAGP ameliorates inflammatory response immediately posttransplant. Proinflammatory cytokines and chemokines locally expressed 24 h and 7 days after transplantation. Concentrations of IL-1β (A and B), IL-6 (C and D), KC/human growth-regulated oncogene (GRO) (E and F), and TNF-α (G and H) were significantly lower in the engrafted islets previously treated with AAGP. Cytokines were measured 24 h and 7 days after transplantation locally to the graft by homogenization (normalized per gram of tissue, n = 3). Data are mean ± SEM adjusted per gram of tissue (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Among the chemokines measured acutely posttransplant, KC secretion, involved in neutrophil recruitment, was significantly overexpressed in Tac+ and, again, significantly reduced in the presence of AAGP (85.4 vs. 32 pg/g tissue, n = 3, P < 0.001). By day 7, the cytokine was clearing, and differences were no longer evident (Fig. 5E and F). TNF-α levels, on the other hand, were not significantly increased on day 1 but became notably different on day 7 (Tac 33.9, Tac+ 76.7, Tac+AAGP 48.7 pg/g tissue; P < 0.001) (Fig. 5G and H).

Intragraft apoptosis showed an increasing trend in cleaved caspase-3 concentration and TUNEL-positive cells in the Tac+ group compared with the rest of the groups, suggesting increased in vivo cell death after CNI treatment and a subsequent AAGP protective effect. However, differences did not reach statistical significance (Tac+ vs. Tac+AAGP: fold change in caspase-3 2.9 vs. 1.9, not significant; TUNEL-positive cells 35.3 ± 26.7% vs. 7.9 ± 8%, not significant) (data not shown).

AAGP Supplementation Improves Human Islet Transplant Function Despite Tac Exposure

The remaining transplanted mice (n = 7 per group) were followed beyond 60 days. Delayed engraftment was observed as expected in this marginal islet mass model. Blood glucose improved over time in the Tac and Tac+AAGP groups, with the proportion of euglycemic animals being significantly higher than those in the Tac+ group, where all mice demonstrated poor function (P < 0.05) (Fig. 6A and B).

Figure 6

AAGP supplementation improves islet transplant function despite Tac exposure. Posttransplant graft function in immunodeficient mice receiving minimal human islet mass (1,000 IEQ). Islets were previously treated with or without Tac/AAGP. A and B: Pooled blood glucose profiles and percent of mice reaching euglycemia demonstrate long-term graft function (60 days) with a nonfunctioning graft for the Tac+ group. Graft-bearing nephrectomy was performed on day 60 to demonstrate graft-dependent euglycemia. A: Horizontal continuous line at 11 mmol/L indicates the normoglycemia limit. Vertical dotted line refers to graft-bearing nephrectomy on day 60, resulting in acute hyperglycemia, thus confirming the islet transplant was responsible for normoglycemia. C: IPGTT to evaluate metabolic response after receiving a glucose bolus. D: The Tac+ group was intolerant to high glucose, which also corresponded to less residual insulin content when grafts were removed after 60 days posttransplant. Data are mean ± SEM adjusted per gram of tissue (Tacn = 6, Tac+n = 3, Tac+AAGP n = 7). *P < 0.05, **P < 0.01, ***P < 0.001. Tx, transplant.

Figure 6

AAGP supplementation improves islet transplant function despite Tac exposure. Posttransplant graft function in immunodeficient mice receiving minimal human islet mass (1,000 IEQ). Islets were previously treated with or without Tac/AAGP. A and B: Pooled blood glucose profiles and percent of mice reaching euglycemia demonstrate long-term graft function (60 days) with a nonfunctioning graft for the Tac+ group. Graft-bearing nephrectomy was performed on day 60 to demonstrate graft-dependent euglycemia. A: Horizontal continuous line at 11 mmol/L indicates the normoglycemia limit. Vertical dotted line refers to graft-bearing nephrectomy on day 60, resulting in acute hyperglycemia, thus confirming the islet transplant was responsible for normoglycemia. C: IPGTT to evaluate metabolic response after receiving a glucose bolus. D: The Tac+ group was intolerant to high glucose, which also corresponded to less residual insulin content when grafts were removed after 60 days posttransplant. Data are mean ± SEM adjusted per gram of tissue (Tacn = 6, Tac+n = 3, Tac+AAGP n = 7). *P < 0.05, **P < 0.01, ***P < 0.001. Tx, transplant.

Thirty days posttransplant, mice underwent IPGTT to evaluate transplant function. The Tac and Tac+AAGP groups both responded appropriately, but Tac+ remained hyperglycemic at 120 min (AUC: Tac vs. Tac+AAGP, 92.6 vs. 91.2, P > 0.05; Tac vs. naive, 92.6 vs. 71.4, P > 0.05; Tac+AAGP vs. naive, 91.2 vs. 71.4, P > 0.05; Tac+ vs. Tac+AAGP, 149.8 vs. 91.2, P < 0.05) (Fig. 6C).

All mice reverted to their previous diabetic state after islet-bearing nephrectomy. Insulin content was assessed as a measure of residual islet mass after 30 days. Figure 6D shows significant differences between groups with reduced insulin content in grafts exposed to Tac. Again, presence of AAGP was beneficial in islet protection despite exposure to Tac (Tac+ vs. Tac+AAGP, 30.9 vs. 100.8 ng/mL, P < 0.01).

AAGP Supplementation Improves Islet Transplant Function Despite Continuous Recipient Treatment With Tac

In further support of the findings, syngeneic diabetic mice were implanted with subcutaneous micro-osmotic pumps to model continuous posttransplant Tac treatment in clinical practice. As with the in vitro findings, transplanted islets exposed to Tac were unable to effectively secrete insulin or return mice to euglycemia during the treatment course. The presence of AAGP, however, restored normal islet function despite Tac exposure, similar to controls (P < 0.001) (Fig. 7A and B).

Figure 7

AAGP supplementation improves islet transplant function despite continuous (short- and long-duration) Tac treatment. Posttransplant graft function in mice receiving syngeneic full mass (500 islets) islet transplant. AAGP was added to the culture media 1 h before transplant, and Tac was administered through a subcutaneous micro-osmotic pump (implanted during the same procedure) at a continuous rate of 1 mg/kg/day. A: Pooled blood glucose profiles of animals over 40 days, with clear dysfunction for Tac+ islets seen during the presence of Tac. The vertical line indicates Tac treatment cessation at day 7 and marks a gradual recovery of Tac+ grafts. The horizontal line at 11 mmol/L indicates the normoglycemia limit. Graft recovery nephrectomy was performed again on day 30. B: Mean time to euglycemia after transplant showing an earlier reversal of diabetes in Tac+AAGP mice (P < 0.001, log-rank [Mantel-Cox] test). Glucose tolerance tests showed a significant difference in graft response for Tac+ when mice received Tac (7 days) and when recipients were CNI free. These differences were not observed in the Tac+AAGP group. C and D: Glucose tolerance tests of mice receiving continuous treatment with Tac showed impairment in the Tac+ group (C), which was fully reversed once the CNI treatment was ceased (D). EH: A similar experiment was conducted with long-duration subcutaneous pumps providing Tac for 28 days. Results show a consistent and significant difference in immediate posttransplant function for mice receiving AAGP-supplemented islets. *P < 0.05. Tx, transplant.

Figure 7

AAGP supplementation improves islet transplant function despite continuous (short- and long-duration) Tac treatment. Posttransplant graft function in mice receiving syngeneic full mass (500 islets) islet transplant. AAGP was added to the culture media 1 h before transplant, and Tac was administered through a subcutaneous micro-osmotic pump (implanted during the same procedure) at a continuous rate of 1 mg/kg/day. A: Pooled blood glucose profiles of animals over 40 days, with clear dysfunction for Tac+ islets seen during the presence of Tac. The vertical line indicates Tac treatment cessation at day 7 and marks a gradual recovery of Tac+ grafts. The horizontal line at 11 mmol/L indicates the normoglycemia limit. Graft recovery nephrectomy was performed again on day 30. B: Mean time to euglycemia after transplant showing an earlier reversal of diabetes in Tac+AAGP mice (P < 0.001, log-rank [Mantel-Cox] test). Glucose tolerance tests showed a significant difference in graft response for Tac+ when mice received Tac (7 days) and when recipients were CNI free. These differences were not observed in the Tac+AAGP group. C and D: Glucose tolerance tests of mice receiving continuous treatment with Tac showed impairment in the Tac+ group (C), which was fully reversed once the CNI treatment was ceased (D). EH: A similar experiment was conducted with long-duration subcutaneous pumps providing Tac for 28 days. Results show a consistent and significant difference in immediate posttransplant function for mice receiving AAGP-supplemented islets. *P < 0.05. Tx, transplant.

Similar findings were observed when using subcutaneous pumps providing Tac treatment for a longer duration (28 days). Again, AAGP-supplemented islets functioned normally and rendered normoglycemia for all animals, despite a single 1-h AAGP treatment of islets before transplant (P < 0.01) (Fig. 7E and F).

Results were corroborated by IPGTTs performed in both treatment modalities (short and long duration). Tolerance tests performed under Tac treatment showed impaired glucose control in the Tac+ group, whereas Tac+AAGP behaved similar to controls (P < 0.001) (Fig. 7C and G).

Tac treatment cessation resulted in normalization of graft function and euglycemia in all animals. Repeat IPGTTs at this stage (30 and 45 days) showed no residual differences between groups (Fig. 7D and H).

We demonstrate that the addition of a potent antifreeze protein AAGP only to the islet culture media for a 48-h exposure affords considerable protection of human islet survival and in vitro function. This protective effect is especially pronounced when used to prevent Tac-induced islet toxicity.

Tac currently is regarded as a mainstay potent immunosuppressant given to prevent both auto- and alloimmunity after clinical islet transplantation (35). Prolonged exposure to CNI class immunosuppressants is strongly associated with nephrotoxicity and posttransplant diabetes in all organ transplants (4).

Tac is known to impair insulin secretion in the native pancreas after pancreas and, especially, islet transplantation and is characterized by impairment of early secretion and decreased biosynthesis. Several associated mechanisms have been defined, including calcineurin/nuclear factor of activated T-cell signaling inhibition (36), insulin gene suppression (37), mitochondrial arrest (38), and decreased posttransplant vascularization (39). In the current experimental model, the addition of Tac resulted in a striking inhibition of insulin secretion and cell death in vitro and impaired islet engraftment and function in vivo. Furthermore, we confirmed islet function impairment after Tac exposure in vitro and found that AAGP was able to reestablish insulin release despite acute exposure to high-dose Tac.

An increased loss of islets during culture was associated with apoptosis observed after in vitro exposure to Tac. Increase in cleaved caspase-3 and TUNEL staining indicated significantly higher cell death in the Tac+ group, but this was diminished after AAGP supplementation.

Islet high susceptibility to hypoxia throughout all stages of cell procurement, preparation, and intraportal transplantation relates to their intrinsic oxygen demand and size, especially that related to islet seeding density in culture (40). Islets are prone to oxidative stress due to decreased antioxidant capacity (41). These elements contribute to islet loss during culture and after transplantation. The current findings confirm an increase in oxidative stress after Tac exposure, with increased extracellular ROS. AAGP supplementation reduced oxidative stress in this model. Similar redox modulation findings have also been noted when using AAGP with other cell lines (17).

In vivo studies complemented all in vitro findings, which demonstrated that AAGP supplementation suppressed early inflammation and improved islet engraftment with long-term efficacy. AAGP-supplemented islets showed significantly reduced expression of IL-1β and IL-6 along with decreased secretion of KC and TNF-α, despite exposure to Tac in culture. These cytokines and chemokines are key participants in the posttransplant inflammatory response and subsequent adaptive immunity activation (42) as well as vital elements in the early clinical posttransplant phase (30). These findings are consistent with previous experiments showing reduced expression of cyclooxygenase-2 expression in HeLa cells exposed to increasing concentrations of IL-1β in the presence of AAGP (17).

Tac exposure was provided by continuous subcutaneous micro-osmotic pump for 7 and 28 days in syngeneic mice. We chose this approach in selected experiments because twice-daily oral gavage of Tac would have been too stressful, but we wished to maintain sustained clinically relevant drug exposure for transplanted islets. We observed that marked impairment of transplanted islets occurred immediately following Tac exposure, which lasted throughout Tac exposure, but this was reversible after withdrawal of Tac. Islets treated with AAGP, however, were protected from Tac toxicity and functioned similar to control in both the short- and the long-duration treatment groups. Because a marked and prolonged posttransplant engraftment and functional benefit was consistently observed when AAGP treatment was confined only to the in vitro culture period, this treatment could potentially be readily applied in clinical studies to enhance islet engraftment and function in patients receiving Tac immunosuppression.

In exploring potential mechanisms of action of AAGP, we found no beneficial effect upon insulin synthesis or storage. Furthermore, we did not find an interactive impact of AAGP on the immunosuppressive properties of Tac. We found that neither Tac nor AAGP affected glucose-stimulated calcium influx in islets, which is a key element in the insulin secretion mechanism of β-cells. This information supports evidence pointing to a potential Tac mechanistic site further downstream in the secretory pathway (43). Conversely, islet capacitance measurements in the current studies revealed significant differences between Tac+ and TAC+AAGP, findings that suggest impaired insulin exocytosis in the presence of Tac, which was reversed by AAGP.

In conclusion, supplementation of islets with AAGP during culture enhanced both the quality and the yield of postculture human islets, which translated into improved engraftment despite the presence of Tac. AAGP also protected islets continuously exposed to Tac posttransplantation, with improved efficacy and decreased inflammatory response. Clinical translation of these findings could potentially offer a means to protect islets both in vitro and in vivo from diabetogenic immunosuppression after transplantation as a means to enhance single-donor islet engraftment and durable long-term function.

Funding. Funding sources include the Diabetes Research Institute Foundation Canada, the Alberta Innovates–Health Solutions (AIHS) Collaborative Research and Innovation Opportunities Team Award, and the Alberta Diabetes Institute. B.L.G.-L. is supported by an Izaak Walton Killam Memorial Scholarship, an AIHS Clinician Fellowship, and by the Canadian National Transplant Research Program. P.E.M. is supported by a Canada Research Chair in Islet Biology. A.M.J.S. is supported through a Canada Research Chair in Transplantation Surgery and Regenerative Medicine and through AIHS as a senior scholar.

Duality of Interest. L.G.Y. owns shares in ProtoKinetix Inc., the company that owns the patent rights to the AAGP molecule. Subsequent to completion of this study A.M.J.S. now serves as a consultant to ProtoKinetix Inc. No other potential conflicts of interest relevant to this article were reported.

Author Contributions. B.L.G.-L. contributed to the design and performance of experiments, data research, and writing of the manuscript. A.R.P. contributed to the performance of experiments, data research, discussion, and review and editing of the manuscript. R.L.P. isolated mouse islets and reviewed the manuscript. D.O. performed D-IP and reviewed the manuscript. T.K. isolated human islets and reviewed the manuscript. A.Br., N.A., and M.B. researched data. A.Ba. performed the capacitance studies. J.E.M.F. performed calcium imaging and reviewed the manuscript. L.G.Y. contributed to the study design, provision of AAGP, and review of the manuscript. P.E.M. contributed to the data research and review of the manuscript. A.M.J.S. contributed to the experimental design, data research, and review and editing of the manuscript. A.M.J.S. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Prior Presentation. Parts of this work were presented orally at the 2015 International Pancreas & Islet Transplant Association, International Xenotransplantation Association, and Cell Transplant Society Congress, Melbourne, VIC, Australia, 15–19 November 2015.

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