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Basic and Experimental Research

GABA Protects Human Islet Cells Against the Deleterious Effects of Immunosuppressive Drugs and Exerts Immunoinhibitory Effects Alone

Prud’homme, Gérald J.1,2,3,7; Glinka, Yelena1; Hasilo, Craig4; Paraskevas, Steven4; Li, Xiaoming1; Wang, Qinghua1,5,6

Author Information
doi: 10.1097/TP.0b013e31829c24be
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Abstract

Islet transplantation has gained acceptance as a treatment modality for type 1 diabetes in some settings (1, 2). However, despite improvements in methodology, islet cell survival remains a major issue. A large portion of the transplanted islet mass is lost rapidly probably due to a combination of hypoxia and damaging innate immune responses (3–5). After that initial phase, there is gradual decline in islet function and many patients revert to hyperglycemia (1, 2). This could be because of persistent anti-islet autoimmunity and chronic allograft rejection, but the toxicity of immunosuppressive drugs toward islet cells appears to be important. It has been shown that rapamycin and tacrolimus (FK506) can impair β-cell function and survival (6–8). Mycophenolate mofetil (MMF) also exerts toxic effects (8). This highlights the need to protect islets in both the short-term and long-term after transplantation.

We recently found that γ-aminobutyric acid (GABA) protects mouse β cells against injury and prevents murine type 1 diabetes (9). Furthermore, it induced islet β-cell regeneration and reversed disease. Importantly, GABA exerted anti-inflammatory and immunosuppressive effects.

It is unclear whether human islets can be protected in the same way. Our objectives were to determine whether human islet and immune cells respond to GABA in a manner similar to mouse cells and to investigate potential protective effects against drugs employed in islet transplantation. We show that GABA improves human islet cell survival in vitro and alleviates the toxic effects of rapamycin, FK506, and MMF. We report that GABA suppresses human immune cells similarly to mouse cells, and it can be combined with rapamycin for an improved immunosuppressive effect. Notably, GABA inhibited nuclear factor (NF)-κB activation in both islets and lymphocytes and interfered with calcium-dependent signaling in murine and human lymphocytes. Because an intracellular increase in Ca2+ levelsis a key early signal in T-cell activation (10–13), this may explain the immunosuppressive effect. Our findingssuggest that GABA might find applications in clinical islettransplantation.

RESULTS

GABA Increases Human Islet Cell Survival in Culture

In preliminary experiments, a GABA concentration of 100 μM was most effective at preventing the apoptosis of human islet cells, and subsequent experiments were performed at that concentration. The effects of GABA in vitro diminished over time (not shown), possibly as a result of degradation or metabolism, and we replenished it in the medium daily in all experiments. Human islets were cultured (5.56 mmol/L glucose) with or without GABA (Fig. 1A–C). Without GABA, human islet cell survival was poor after 7 days, with a markedly decreased number of live cells. However, the addition of GABA resulted in an approximately threefold increase in the number of surviving cells by day 7.

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FIGURE 1:
GABA increases the survival of human pancreatic islets in culture and stimulates insulin secretion. Islets were cultured in CMRL 1066 medium supplemented with 10% heat-inactivated fetal bovine serum and glutamine for 6 to 7 days with daily refreshment of the medium and GABA. Cell viability was tested by Annexin V and PI staining assay by flow cytometry. A, GABA (100 μM) increased the total number of live cells per islet sample, identified based on PI-/Annexin V- (double-negative) staining (P<0.02). Data represent the number of total live cells per sample, calculated as described in Materials and Methods. Each sample was in triplicate and the experiment was repeated with two independent islet donors. B, GABA (100 μM) decreased the fraction of apoptotic islet cells (P<0.05). Apoptotic cells were identified based on Annexin V+ staining and can be subdivided into early apoptotic cells (PI-/Annexin V+) or late apoptotic cells (PI+/Annexin V+). Each sample was in triplicate and the experiment was repeated with two independent islet donors. C, an example of the flow cytometry data. Human islets were cultured for 2 days in the absence or the presence of 100 μM GABA. GABA reduced apoptotic cells and increased live cells. D, GABA significantly increased insulin secretion by human islets. Human islets were incubated (or not) with 100 μM GABA at 2 or 11 mmol/L glucose (as described in Materials and Methods), and insulin levels were measured by radioimmunoassay. Data represent mean±SE and are representative result of two independent experiments done in triplicate. *P≤0.05.

We analyzed the cells for apoptosis by propidium iodide (PI) and Annexin V staining by flow cytometry (Fig. 1B,C). PI staining identifies dead cells, except early apoptotic cells, whereas Annexin V staining identifies cells that are undergoing apoptosis at early and late stages. The proportion of apoptotic cells, as detected by Annexin V staining, was significantly decreased by GABA.

GABA Increased Insulin Secretion in Human Islets

Human islets were incubated at either 2 or 11 mmol/L glucose in serum-free conditions for 30 min followed by the addition of GABA (100 μM) for an additional 30 min. This is a short-term assay where differences in cell survival are unlikely to occur. At both glucose concentrations, GABA significantly increased insulin secretion (Fig. 1D).

GABA Protects Against the Toxicity of Immunosuppressive Drugs

We added graded concentrations of either rapamycin, FK506, or MMF to the human islet cultures, with or withoutGABA (100 μM). As shown in Figure 2A–F, these immunosuppressive drugs all increased the proportion of apoptotic cells (Annexin V+) and reduced the proportion of live cells (PI-/Annexin V-) after 2 days in culture. However, when GABA was added, in all cases, the number of live cells was significantly increased, whereas the number of apoptotic cells was significantly decreased.

F2-6
FIGURE 2:
Treatment of human islets with GABA protects against the detrimental effects of conventional immunosuppressive drugs. GABA increased the proportion of live cells and reduced the proportion of apoptotic cells in the immunosuppressive drug-treated samples (A–F). The islets were cultured for 2 days with rapamycin (A and B), FK506 (C and D), or MMF (E and F) with or without 100 μM GABA. The medium and drugs were refreshed daily. Live cells were identified by flow cytometry based on PI-/Annexin V- (double-negative) staining. Data in A, C, and E represent the percentage of live cells. The difference induced by GABA between the groups in B, D, and F was statistically significant (P<0.05) with all three drugs (rapamycin, FK506, and MMF). Each sample was in triplicate and the experiment was repeated with two independent islet donors. Apoptotic cells correspond to Annexin V+ cells (sum of right top and bottom quadrants) in each flow cytometry panel, as shown in B, D, and F. Representative flow cytometry data are presented with 5 nM rapamycin (B), 250 nM FK506 (D), and 10 nM MMF (F).

GABA Suppresses NF-κB Activation

We noted activation of canonical NF-κB in cultured human islets, and the addition of GABA suppressed activation of this pathway (Fig. 3A). This GABA-mediated effect was blocked by a GABA type A receptor (GABAAR) antagonist (picrotoxin) but not by a GABA type B receptor (GABABR) antagonist (saclofen). Thus, GABA appears to inhibit NF-κB by activating GABAAR only. Because NF-κB is a key pathway in immunity (14), we examined immune cells. GABA suppressed NF-κB activation in anti-CD3–activated (Fig. 3B) or concanavalin A–activated (not shown) human peripheral blood mononuclear cells (PBMCs). Both these stimuli activate T cells. We also stimulated mouse splenic T cells with combined anti-CD3/CD28 monoclonal antibody (mAbs; a powerful T-cell stimulant), and again, we observed that GABA suppressed NF-κB activation (Fig. 3C).

F3-6
FIGURE 3:
GABA suppresses activation of NF-κB in human islets cells and human or mouse lymphocytes. A, human islets were incubated with or without 100 μM GABA in the culture medium for 2 hr in triplicates. The GABAAR antagonist picrotoxin (100 μM) or GABABR antagonist saclofen (100 μM) were added or not, and NF-κB activation was determined (see Materials and Methods). Data are expressed as a percentage of NF-κB activation in the nontreated cells and demonstrate that picrotoxin rather than saclofen reversed the inhibitory effect of GABA. The results are representative of the two independent experiments. B, human donor PBMCs were stimulated with matrix-bound anti-human CD3 antibody (2 hr) with or without GABA (100 μM). GABA significantly suppressed NF-κB activation. The results are representative of the two independent experiments. C, mouse splenic T cells were stimulated with matrix-bound anti-mouse CD3 and CD28 antibodies for 2 hr with or without GABA (100 μM). GABA significantly suppressed NF-κB activation. The results are representative of the two independent experiments. For A to C, **P≤0.01, ***P≤0.001, or ****P≤0.0001.

GABA Blocks Calcium Influx in CD3-Stimulated T Cells

T-cell activation is accompanied by a transient increase in cytosolic Ca2+. We stimulated human PBMCs (Fig. 4A,B) and mouse splenic T cells (Fig. 4C) with anti-CD3 mAb and measured changes in intracellular Ca2+ levels. GABA (100 μM) blocked the transient increase in Ca2+, which was induced by anti-CD3, and was as effective as a conventional calcium channel–blocking drug (nifedipine) (Fig. 4A). This inhibition appears to be mediated through the GABAAR, because it was abolished by picrotoxin (Fig. 4B). This activity of GABA was similarly observed in mouse T cells (Fig. 4C).

F4-6
FIGURE 4:
GABA blocks the increase in intracellular Ca2+ levels induced by anti-CD3. A, GABA (100 μM), like nifedipine (1 μM; L-type calcium channel blocker), prevents Ca2+ influx in human PBMCs activated by soluble anti-CD3 antibody (2 μg/mL). Data represent relative Ca2+ levels 1 min after the addition of anti-CD3. At 10 min, calcium levels had decreased in all samples (not shown), consistent with a transient increase of Ca2+ after T-cell activation. B, inhibitory effect of GABA on human lymphocytes is reversed by 100 μM picrotoxin (GABAAR antagonist). C, mouse splenic T cells are inhibited by GABA similarly to human T cells (A). Data are representative of three independent experiments. For A to C, **P≤0.01, ***P≤0.001, or ****P≤0.0001.

GABA Can Be Combined with Rapamycin for Increased T-Cell Suppression

We activated human PBMCs (Fig. 5A,B) or mouse Tcells (Fig. 5C) with CD3 mAbs. GABA suppressed proliferation, and this was blocked by picrotoxin but not saclofen, consistent with a GABAAR response. GABA did not interfere with rapamycin-mediated suppression, but rather increased it (Fig. 5B,C). In both species, GABA significantly increased suppression at a low rapamycin concentration (eg, 0.01 nM), whereas it was not different at higher rapamycin concentrations (10 or 100 nM).

F5-6
FIGURE 5:
GABA suppresses the proliferation of lymphocytes and increases the suppressive effect of rapamycin. Lymphocytes were activated with matrix-bound anti-CD3 antibodies for 72 hr in quadruplicates. A, picrotoxin (GABAAR antagonist) reverses the inhibitory effect of GABA, whereas saclofen (GABABR antagonist) does not. Human (B) and mouse (C) lymphocytes were treated with the increasing concentrations of rapamycin in the presence or absence of 100 μM GABA for 72 hr in quadruplicates. GABA increased the suppressive activity of rapamycin at low rapamycin concentrations but not at a high rapamycin concentration. Proliferation is expressed as a percentage to the proliferation of the stimulated nontreated cells (no rapamycin, no GABA). Data are representative of three independent experiments. For A to C, ***P≤0.001 or ****P≤0.0001.

DISCUSSION

Clinical islet transplantation reverses disease in most patients, but long-term graft survival remains a problem. Investigators have studied various approaches to improve islet viability, notably the administration of caspase inhibitors (15–17). However, these drugs may have adverse effects, and the safety of prolonged clinical application remains unknown. We have previously shown that GABA protects mouse islet β cells against injury, increases insulin production, and induces regeneration in vivo (9). In this study, we examined whether GABA could exert protective effects on human islet cells. We found that GABA improved survival of human islet cells in vitro, such that the yield of live cells was approximately tripled after 7 days in culture. GABA also significantly increased insulin secretion under culture conditions, where differences in islet cell survival would not be a factor.

Increased cell numbers might have resulted in part from islet cell proliferation because we have observed this to occur (data not shown), but it is insufficient to account for this difference in only 7 days of culture. Here, we show that GABA reduced apoptosis in culture. Furthermore, when we added the antiproliferative agent rapamycin in culture, GABA still increased cell survival considerably. Indeed, GABA increased islet cell survival in the presence of rapamycin, FK506, and MMF. Thus, GABA appears to have a general effect that is not linked to the specific mechanism of immunosuppression.

The ability to reduce the toxicity of immunosuppressive drugs would be of limited utility if their suppressive effects were blocked. However, in vitro, when we combinedGABA with rapamycin, a major component of the Edmonton protocol (18), we did not observe any interference with T-cell suppression; indeed, suppression was improvedat low rapamycin concentrations. This might occurbecause GABA and rapamycin suppress different pathways of activation in T cells, and this question merits further investigation.

The NF-κB pathway is critical to the stimulation of both innate and adaptive immunity (14). However, in islet cells, there is evidence that it is linked to inflammation-induced and postisolation islet cell injury (19–21). Interestingly, we found that GABA suppresses NF-κB activation in both islet cells and lymphocytes. This suggests that it willhave beneficial effect by limiting inflammation-related damage to islet cells while acting as an immunosuppressive agent in the context of islet allograft rejection and autoimmunity.

The mechanisms by which GABA protects islet cells are still unknown. Pancreatic islet cells, including β cells, express the ion channel receptor GABAAR and the G-protein–coupled receptor GABABR (22, 23). The type A receptor is a pentameric ligand-activated chloride channel, which allows movement of Cl- in or out of the cells. It is constituted of various combinations of several subunits (usually 2 α, 2 β, and a third subunit) and multiple type A receptors exist. The type B receptor has only two subunits and is not variable. Ligand binding to these receptors results in the activation of several signaling pathways, some of which (e.g., phosphatidylinositol 3-kinase/Akt and extracellular signal-regulated kinase) have been linked to increased cell survival. However, as shown in this study, an alternative reason might be the inhibition of NF-κB activation. This will curtail inflammatory pathways that have long been known to be injurious to β cells (see below).

There have been some contradictory reports on the effects of GABA on insulin production by islet cells, with some authors showing stimulatory effects like us (24, 25), whereas others showed inhibitory effects (23, 26) or no effects (27). Some studies were performed in rodents (24, 25, 27), and there might be species differences; however, our own results in mice (9) and humans (this article) are similar. Recently, Braun et al. (25) showed by patch-clamp experiments that GABA depolarizes human β cells and is an autocrine excitatory transmitter for these cells. Interestingly, they found that β cells secreted GABA by glucose-dependent exocytosis of insulin-containing granules and also by a glucose-independent process. GABA depolarized β cells and stimulated action potential firing in β cells in response to glucose. A GABAAR antagonist suppressed insulin secretion induced by glucose (6 mmol/L). These observations are similar to ours in mouse β cells (9). However, we have reported that GABA-modulated insulin secretion is glucose concentration dependent in insulinoma cells, and stimulatory effects were observed at low glucose concentrations but not at very high concentrations (28). Moreover, based on our previous work (29), autocrine insulin can down-regulate GABA-GABAAR signaling and this appears to be a feedback mechanism for fine-tuning β-cell secretion. Whether these findings are relevant to human β cells is unclear, but the current study was conducted at glucose levels ranging from 2 to 11 mmol/L, with stimulatory effects on insulin secretion in that range.

In the case of immune cells, there have been several reports of GABA-mediated inhibitory effects (30–39). We observed that GABA suppresses murine CD4+ and CD8+ T cells as well as macrophages (9). Suppression is not complete, but usually in the range of 30% to 50%. The mechanism of suppression has remained unclear, but Alam et al. (37) reported that GABA reduced calcium influx in formyl-methionyl-leucyl-phenylalanine–activated human PBMCs. This reduction in Ca2+ appeared to be occurring in lymphocytes; however, they did not specifically stimulate T cells. Here, we found that GABA blocks activation-induced increase in intracellular Ca2+ levels in both murine and human T cells. T cells were stimulated through the T-cell receptor complex with anti-CD3 mAb or with the T-cell mitogen concanavalin A. In both cases, we found a severe inhibition of calcium influx. GABA also reduced intracellular Ca2+ levels in unstimulated lymphocytes in culture. Because calcium influx is one of the most important events in T-cell activation (10, 11), this might explain at least in part its immunosuppressive activity. Calcium signaling blockade would prevent many downstream signaling events. How this blockade occurs warrants further investigation. T cells express GABAAR receptors, whereas the GABABR is not expressed (31–33). GABA-mediated inhibition of calcium influx in lymphocytes is in marked contrast to islet cells, where we observed increased calcium levels (9). This undoubtedly reflects profound physiologic differences between these cell types. Because lymphocytes have lower intracellular Cl- levels (compared to islet cells), the activation of the receptor perhaps has a different effect on membrane polarization. We hypothesize that this affects voltage-dependent calcium channels such that calcium influx is blocked.

The immunosuppressive drugs cyclosporine A and FK506 also block calcium signaling by interfering with calcineurin function (40). This highlights the importance of this pathway. On the contrary, rapamycin acts by binding to the mammalian target of rapamycin and inhibiting numerous downstream signaling pathways involved in proliferation and other events (41). Our observations suggest that GABA and rapamycin do not interfere with each other. Indeed, they appear to collaborate, which is likely to be beneficial in the context of clinical islet transplantation.

GABA has been administered orally to humans in large doses (several grams per day), and it was absorbed through the gastrointestinal tract causing either no or only minor adverse effects (42). There have been very few studies of its effects on human endocrine pancreatic function. However, in small studies, GABA or mimetic drugs have reduced blood glucose and increased insulin and C-peptide levels (43, 44). Thus, these human studies suggested that GABA plays a role in regulating endocrine pancreatic function.

In conclusion, our in vitro experiments showed that GABA exerted protective effects on human islet cells, as it does on mouse islet cells. It reduced islet cell apoptosis induced by conventional immunosuppressive drugs. It inhibited NF-κB activation, which has been linked to islet cell injury and immune-cell activation. Thus, it was immunosuppressive on its own. This amelioration of cell survival might be useful for increasing β-cell numbers before islet transplantation. Importantly, GABA counteracted some negative effects of rapamycin on islet cells and did not impair its inhibitory effects on T cells but rather increased suppression. We conclude that GABA has an activity profile that can ameliorate β-cell survival under a variety of conditions. Further studies are required to confirm that these in vitro effects also occur in vivo.

MATERIALS AND METHODS

Human Islets Isolation and Assays

Human islets were isolated as described (19). Pancreata from deceased human donors were retrieved after consent was obtained by Transplant Quebec (Montreal, Quebec, Canada). The pancreas was intraductally loaded with cold CIzyme (Collagenase HA; VitaCyte, Indianapolis, IN) and neutral protease (NB Neutral Protease; SERVA Electrophoresis, Heidelberg, Germany) enzymes, cut into pieces, and transferred to a sterile chamber for warm digestion at 37°C in a closed-loop circuit. Dissociation was stopped using ice-cold dilution buffer containing 10% normal human AB serum, once 50% of the islets were seen to be free of surrounding acinar tissue under dithizone staining. Islets were purified on a continuous iodixanol-based density gradient (Optiprep; Axis-Shield, Dundee, UK) using a COBE 2991 Cell Processor (Terumo BCT, Lakewood, CO). Yield, purity, viability, and glucose-stimulated insulin secretion assays were determined.

Islets were cultured in CMRL 1066 medium (5.56 mmol/L) with 10% heat-inactivated fetal bovine serum and glutamine in ventilated Eppendorf tubes. To study immunosuppressive drugs, the islets were cultured for 48 hr with or without rapamycin, FK506, or MMF, in triplicates. For the analysis of survival, the islets were dispersed into single cells by trypsinization with TRYPLE (Invitrogen, Carlsbad, CA) for 5 min at 37°C and pipetting. These cells were stained with Annexin V-PI kit (Invitrogen), and the number of viable and apoptotic cells was evaluated by flow cytometry. Total number of the viable cells per sample was calculated as follows: (total number of cells per sample)×A, where A is the fraction of the viable cells calculated from the flow cytometry data. A=(% gated viable cells)×(number of gated cells)/(total number of cells).

Insulin Secretion Radioimmunoassay

Human islets were placed into 48-well plates in serum-free Hank’s buffer containing 2 or 11 mM glucose for 30 min at 37°C, and then GABA (100 μM) was added (or not) for an additional 30 min, and insulin levels were measured by human insulin radioimmunoassay kit (Millipore, Billerica, MA).

Immune Cell Isolation and Activation

Human PBMCs were kindly provided by Dr. John Marshall (St. Michael’s Hospital, Toronto, Ontario, Canada) and were obtained from healthy volunteers through an approved protocol of St. Michael’s Hospital Research Ethics Board. Murine T cells were isolated from the spleen and purified by magnetic sorting using a kit from R&D Systems (Minneapolis, MN). They were activated with matrix-bound anti-CD3 and anti-CD28 antibodies, as we have described (9), or with soluble anti-CD3. Human PBMCs were activated with either matrix-bound or soluble anti-CD3 (OKT3) antibody or concanavalin A (5–10 μg/mL).

Proliferation Assay

Cells were plated onto a 96-well plate at 105 cells per well in RPMI 1640 with 10% heat-inactivated serum and 5 × 10-5 M β-mercaptoethanol and cultured for 72 hr. The plate was precoated as described above. Immune cell proliferation was evaluated using WST-1 reagent (Roche, Indianapolis, IN) according to the protocol provided by the manufacturer.

Nuclear Factor-κB

Activation of NF- κB was measured using an enzyme-linked immunosorbent assay from StressGen (San Diego, CA). Protein extracts from the cells were deposited in 96-well plates coated with a DNA oligonucleotide carrying the NF-κB consensus binding sequence. The attachment of active NF-κB was detected with an antibody against the NF-κB p65 subunit. Activation is calculated as relative luminescent units per milligram of protein in the extract and expressed as a percentage of the nontreated cells.

Cytosolic Calcium in Lymphocytes

Intracellular Ca2+ levels were measured using FURA-2AM as described (45). Lymphocytes were preloaded with 10 μM FURA-2AM and treated or not with the antagonist of GABAAR (picrotoxin), L-type Ca2+ channel antagonist nifedipine, and GABA in triplicates at 105 cells per well. To activate lymphocytes, soluble anti-CD3 antibody was added 1 min before data acquisition to determine the Ca2+ level. The time required for activation was optimized in a separate experiment with the acquisition repeated with 1 min intervals within 30 min. The excitation spectra were recorded at 340 and 380 nm and emission at 510 nm at 37°C. The levels of intracellular Ca2+ were calculated as described in (45) and were presented as a percentage of the level in the nontreated cells.

Statistical Analysis

Statistical analysis was performed with GraphPad Prism 6 program (GraphPad Software, La Jolla, CA). Data are presented as mean±SE, the difference between groups was analyzed by analysis of variance, and P<0.05 was considered significant.

ACKNOWLEDGMENTS

The authors acknowledge Dr. John Marshall and Ms. Jean Parodo for providing human lymphocytes for these studies, Dr. Alan Lazarus for providing reagents, and Dr. Indri Nuryani Purwana for data analysis.

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Keywords:

GABA; Islet; Apoptosis; Rapamycin; NF-κB; Immunosuppression

© 2013 by Lippincott Williams & Wilkins