Targeting Uncoupling Protein-2 Improves Islet Graft Function

Abstract

Preserving and enhancing the primary function of transplanted islets is not only crucial for improving the outcome of the islet transplantation, but is also important for reducing the islet mass required to achieve insulin independence. Uncoupling protein 2 (UCP2) is a member of the uncoupling protein family, which is localized to the inner mitochondrial membrane and negatively regulates insulin secretion in the pancreatic β-cells. In this study, we assessed the importance of UCP2 in improving islet graft primary function by using UCP2 gene-knockout (UCP2-KO) mice in a syngeneic islet transplantation model. Islets were isolated from UCP2-KO or wild-type (WT) C57BL/6J mice. The effects of deficiency of UCP2 on islet transplantation and islet function were determined. Two hundred islets from UCP2-KO, but not from WT, donors were capable of completely restoring normoglycemia in 1 week in all syngeneic diabetic recipients. Islets harvested from UCP2-KO mice secreted onefold more insulin in GSIS assay than that from WT mice, and maintained normal GSIS after 72-h exposure to high glucose challenge. In addition, UCP2-KO islets expressed twohold higher Bcl-2 mRNA than that from WT islets, and were resistant to high glucose and proinflammatory cytokine induced death. Our study explored a potential mechanism that may explain the benefit of UCP2-KO islets in islet transplantation. Targeting UCP2 may provide a novel strategy to improve primary function of transplanted islets and reduce the number of islets required in transplantation.

Introduction

Diabetes mellitus is now fast emerging as one of the biggest health catastrophes in modern society. It was estimated that approximately 160 million people worldwide suffered from diabetes in 2000, and this number was projected to increase to 221 million in 2010 and to 366 million in 2030 (25).

Recent progress in the pancreas' enzymatic digestion process along with novel immunosuppression strategies has led to successful clinical trials of islet transplantation in humans (18). However, successful islet transplantation depends on the infusion of higher mean islet mass (>10,000 IE/kg) prepared from 2–4 donor pancreases to achieve insulin independence; the scarcity of islets donors severely limits the number of transplants that can be performed in diabetic subjects because only a small fraction of implanted islets can survive and become engrafted (19).

Transplanted islets are particularly vulnerable in the immediate posttransplantation period (7). Recent study indicated recipient hyperglycemia rendered islet grafts susceptible to dysfunction and failure. An increased incidence of primary nonfunction was observed when a marginal number of islets were transplanted into severely diabetic mice, in comparison with moderately diabetic mice (12). Hyperglycemia increased oxidative stress and deteriorated β-cell function in transplanted islets. Islet graft response to transplantation injury includes upregulation of protective as well as apoptotic genes (17). Therefore, preserving and enhancing the primary function of transplanted islets are not only crucial for improving the outcome of the islet transplantation, but also important for reducing the islet mass required to achieve insulin independence, especially in the current limited sources of islets.

Uncoupling protein 2 (UCP2) is a member of the uncoupling protein (UCP) family, which is localized to the inner mitochondrial membrane; it is the only known uncoupling protein expressed in pancreatic β-cells to date (4). Activated UCP2 may compete with ATP syntheses for the electrochemical energy of mitochondria and result in decreased cellular ATP/ADP levels. Consequently, UCP2 negatively regulates pancreatic β-cell insulin secretion (22). Increasing experiment evidence suggests that the impairment of glucose-stimulated insulin secretion (GSIS) plays an important pathophysiological role in the development of β-cell dysfunction. It has been shown that downregulation of UCP2 improves GSIS under pathophysiologyical conditions (9,11,12). Taken together, these findings suggest that pancreatic β-cell UCP2 is a potential target for improving islets function. To date, the role of UCP2 on mitochondrial ATP production and β-cell insulin secretion has been studied in type 2 diabetes mouse models (6,22). In the present study, we assessed the importance of UCP2 in improving islet graft primary function by using UCP2 gene-knockout mice in a syngeneic islet transplantation model.

Materials and Methods

Experimental Animals

UCP2 gene knock-out (UCP2–KO, C57BL/6J) and UCP2 wild-type (WT, C57BL/6J) mice were provided by Dr. Bradford B. Lowell (Beth Israel Deaconess Medical Center, Boston, MA). All mice were raised and maintained in the animal facilities of University of Pittsburgh. Mice at 8–10 weeks of age and with body weight around 25 g were used in the experiment. All animal protocols are approved by the Institutional Animal Care and Usage Committee at the University of Pittsburgh.

Islet Isolation and Culture

The islet isolation of UCP2- KO and WT C57BL/6J mice was conducted in parallel as described previously (24). Briefly, the pancreas was injected through the pancreatic duct with Hanks' buffered saline solution (HBSS, Mediatech, Herndon, VA) containing collagenase type V (Sigma, St. Louis, MO), excised, digested for 10–15 min at 37°C water bath, and then filtered through a 500-μm wire mesh. The digested pancreas was rinsed with HBSS, and islets were separated by Ficoll gradient centrifugation. After several washes in HBSS, islets were handpicked and maintained in RPMI-1640 (Mdeiatech) containing 11 mM glucose.

Streptozocin (STZ) Treatment

Eight- to 10-week-old male WT C57BL/6J mice with body weight around 25 g were used as islet graft recipients that were rendered diabetic with a single intraperitoneal injection (250 mg/kg) of STZ (Sigma). An animal was considered diabetic after two consecutive blood glucose readings >300 mg/dl.

Islet Transplantation and Graft Function Evaluation

Islet transplantation was performed as previously described (24). Briefly, the WT recipients were anesthetized with 100 mg/kg ketamine and 10 mg/kg xylazine. Under sterile conditions, the kidney was externalized through a small incision in the flank. A total of 300 or 200 fleshly isolated islets of similar size (~150 μm in diameter) from UCP2-KO or WT donors were aspirated into PE50 tubing, and then inserted under the renal capsule. Islet graft function was monitored by serial blood glucose measurements through tail vein bleeding from fasting mice (Accu-Check III blood glucose monitor, Biehringer Mannheim, Idianapolis, IN). Primary graft nonfunction was defined as the inability to reach nonfasting blood glucose levels less than 200 mg/dl for two consecutive measurements 5 days posttransplantation (13).

Glucose Tolerance Test (GTT)

Mice were fasted overnight and injected intraperitoneally with glucose solution at a dose of 1 g/kg body weight. Blood glucose levels were measured before and at 10-min intervals after glucose injection.

Islets Glucose-Stimulated Insulin Secretion (GSIS)

After being isolated as above, islets were incubated in RPMI-1640 containing 11 mM glucose (normoglycemia condition) or RPMI-1640 containing 25 mM glucose (hyperglycemia condition). The procedure was followed as previously described (21) with some modifications. Before assay, groups of 10 islets of similar size were transferred to microcentrifuge tubes, pelleted, and the medium aspirated and preincubated in Krebs-Ringer's Buffer (KRB) with 3 mM glucose for 1 h at 37°C. After washing, they were incubated in fresh KRB containing either low glucose (3 mM) or high glucose (20 mM) for 30 min. Supernatant from each well was collected after each 30 min incubation (first 3 mM and then change to 20 mM), and the concentration of insulin was measured using rat/mouse insulin ELISA kit (Linco Res. Inc). Insulin secretion was expressed as percentage of insulin content. Islet DNA was measured using the CyQUANT cell proliferation assay kit (Molecular Probes).

Assessment of Islet Cell Viability by Confocal Imaging Analysis

The viability of islets was determined by a semiquantitative measurement using propidium iodide (PI) staining, a vital dye that only permeates cells with damaged membranes. The percentage of dead cells in the islets is approximated by visual enumeration (1). Isolated islets were exposed to the 25 mM high glucose and three inflammatory cytokines IL-1β (5 ng/ml), IFN-γ (100 ng/ml), and TNF-α (10 ng/ml) for 24 h (all from Roche Diagnostics). Then the islets were double stained with PI and Hoechst 33342 nuclear dyes (Sigma) before imaging analysis using confocal fluorescent microscope.

Quantification of Gene Expression

For real-time RT-PCR analysis, total RNA was extracted from whole islets and recovered islet grafts using RNeasy Plus Mini (Qiagen). First-strand cDNA was synthesized from total RNA by RT reaction using SuperScript First-Strand Synthesis SuperMix (Invitrogen). The quantitative real-time PCR analysis was performed on Real-time-PCR system 7500 (Applied Biosystems, Foster City, CA) using the Assay-on-Demand TaqMan probes (Mm00627598_m1 for UCP2, Mm00477631_m1for Bcl-2, all from Applied Biosystems).

Statistical Analyses

Statistical analyses were performed using a two-tailed Student's t-test and two-way ANOVA with Bonferroni post hoc test. A value of p < 0.05 was considered significant.

Results

UCP2 Deficiency Improves Primary Function of Pancreatic Islet Grafts

To determine the effects of UCP2 deficiency on primary function of islet grafts we utilized a syngeneic islet transplantation model. The C57BL/6J recipients were rendered diabetic by single injection of STZ and were randomly assigned to two groups. Because our preliminary data showed that 300 islets from both UCP2-KO and WT C57BL/6J donors were capable of restoring the normal glycemia on day 3 posttransplantation in all diabetic syngeneic WT recipients (Fig. 1A), we utilized 200 islets in this study to assess islet function after transplantation into syngeneic recipients. Aliquots of 200 freshly isolated islets of similar size (~150 μm in diameter) from UCP2-KO or WT donors were transplanted under the renal capsule of diabetic recipients (478 ± 10.2 mg/dl). As show in Figure 1B, diabetic mice transplanted with 200 islets from UCP2-KO mice were completely restored to normoglycemia (146.8 ± 5.1mg/dl) in 1 week. In contrast, diabetic mice receiving the same number of WT islets had delayed primary graft function up to 22 days posttransplantation. Also, we removed the kidney bearing the islet grafts of three recipients from each group after 3 months of islet transplant; all became diabetes again in two days after nephrectomy (data not shown). It suggests that there was no presence of residual function of islets in the pancreas after STZ injection.

Figure 1.

UCP2 deficiency improves pancreatic islets graft function. (A) Blood glucose values were assessed before and every day post-islet transplantation (n = 6 per group) (p > 0.05). (B) Blood glucose values were assessed before and every 4 days post-islet transplantation (n = 6 per group) (p < 0.005). (A) and (B) analyzed by Kaplan-Meier Log-Rank. (C) Glucose tolerance test. At day 12 posttransplantation syngeneic islet graft recipients were fasted overnight, followed by injection of 1 g/kg glucose, IP (Time 0). Blood glucose levels were assessed before and after glucose injection at 10-min intervals (n = 6 per group). Area under the curve (AUC) is 33,100 versus 43,257. Black squares: UCP2-KO group, gray triangle: WT group. Results are expressed as mean ± SEM. UCP2-KO versus WT: ^*p < 0.05 and ^**p<0.01; unpaired, two-tailed Student's t-test.

To further study the effects of UCP2 deficiency on islet graft function, glucose tolerance tests were performed at day 12 posttransplantation (Fig. 1C). Following intraperitoneal infusion of glucose (1 g glucose/kg mouse weight), the recipients receiving UCP2-KO islets exhibited normal glucose profiles. After 20 min of glucose load, blood glucose levels were elevated to 519.2 ± 29.2 mg/dl, followed by a decline to prechallenge levels within 2 h. In contrast, the elevated glucose levels of recipients receiving WT islets followed glucose load declined slowly and remained at 245.0 ± 25.1 mg/dl upon 2-h post-glucose challenge, a impaired pattern of GTT (Fig. 1C). Area under the curve (AUC) is 33,100 versus 43,257.

UCP2 Gene Expression Is Increased by Glucose Challenge In Vitro

To assess the effect of high glucose on UCP2 gene expression in islets, we performed real-time quantity PCR. As expected, no UCP2 mRNA expression was detected from UCP2-KO islets. The UCP2 mRNA levels from WT islets that were exposed to 25 mM glucose for 72 h was significantly increased 2.3-fold in comparison with base level expression (p < 0.05) (Fig. 2). This is consistent with previous reports that both UCP2 protein levels and mRNA were upregulated by hyperglycemia (11,15).

Figure 2.

High glucose challenge upregulated UCP2 gene expression in WT islets. UCP2 mRNA expression levels in WT islets cultured for 72 h at 25 mM high glucose were measured. Expression levels are expressed as relative to the control group (before culture). Results are expressed as mean ± SEM from three independent repeats with six replicates. UCP2 gene relative to an arbitrary value of 1, which was assigned to the expression levels in control animals. ^*p < 0.05, unpaired, two-tailed Student's t-test.

UCP2-KO Pancreatic Islets Have Increased Insulin Secretion

Previous studies indicated that UCP2 negatively regulates insulin secretion. We investigated the direct effect of UCP2 deficiency on islets' insulin secretion. Pancreatic islets were isolated from UCP2-KO and wild-type control mice, cultured 24 or 72 h in 11 mM glucose, and then assessed GSIS. As shown in Figure 3A and B, UCP-KO islets secreted 1.5-fold higher insulin under high glucose stimulation than that from WT mice (1.86 ± 0.2% vs. 0.80 ± 0.2% of insulin content, respectively, after 24-h culture; p < 0.001). These data are consistent with other reports showing that UCP2 deficientcy enhanced the islets' GSIS (9,22). The insulin content was normalized with DNA content (Table 1).

Figure 3.

UCP2-KO islets had increased insulin secretion in response to the stimulation of high glucose and are more potent after exposed to high glucose challenge both in short term and long term. (A) Islets isolated from the knock-out (UCP2-KO) or wild-type (WT) mice were incubated with 11 mM glucose in RPMI-1640 medium for 24 h. After washing, islets were exposed to 3 mM glucose in Krebs-Ringer's Buffer and then 20 mM glucose in Krebs-Ringer's Buffer for 30 min each. Insulin secretion in response to varying glucose was measured (GSIS). (B) Islets were exposed to 25 mM glucose in RPMI-1640 medium for 24 h and washed, then GSIS was performed. (C) Islets were exposed to 25 mM glucose in RPMI-1640 medium for 72 h and washed, then GSIS was performed. (D) Islets were exposed to 11 mM glucose in RPMI-1640 medium for 72 h and washed, then GSIS was performed. Results are expressed as mean ± SEM from three independent repeats with each condition carried out in six replicates and 10 islets in each replicate. The results were normalized to total insulin content and the number of islets present. ^**p < 0.001 UCP2 versus WT; ^#p < 0.05 and ^##p < 0.001 25 mM versus 3mM; NS: no significant difference; two-way ANOVA with Bonferroni posttests.

Table 1.

Insulin and DNA Content of UCP-KO and WT Islets Incubated in Different Conditions

	11 mM 24 h	25 mM 24 h	11 mM 72 h	25 mM 72 h
Insulin content (ng insulin/islet)
UCP-KO	284.1 ± 14.5	357.2 ± 15.5	300.7 ± 17.1	448.3 ± 20.6
WT	297.0 ± 21.1	383.0 ± 20.8	311.1 ± 25.7	429.7 ± 16.4
DNA content (ng/islet)
UCP-KO	6.2 ± 1.5	7.3 ± 0.8	6.7 ± 0.9	8.9 ± 1.7
WT	6.4 ± 2.0	8.1 ± 1.1	6.6 ± 1.8	9.2 ± 1.5

This table was used to calculate the GSIS data present in Figure 3. Before islets culture, insulin content relative to DNA content was similar in UCP2-KO versus control wild-type (WT) (data not shown). High glucose (25 mM) incubation increased both insulin and DNA content in islets from UCP-KO and WT mice in a time-dependent manner, but insulin content relative to DNA content was unchanged.

The Islets From UCP2-KO Mice Are More Potent After Exposed to High Glucose Challenge Both in Short Term and Long Term

Studies have shown that chronically elevated glucose levels can cause β-cell dysfunction (11). The transplanted islets exposed to sustained hyperglycemia in diabetic recipients have shown impaired β-cell function (10). Then, we further tested whether UCP2–KO islets were more resistant to hyperglycemia-induced GSIS impairment. We incubated WT and UCP2-KO islets in RPMI-1640 medium containing 25 mM glucose for 24 and 72 h and then assessed GSIS (Fig. 3C, D). Following high glucose culture, islets increased basal insulin release in both groups in a time-dependent manner. WT islets had elevated basal insulin secretion from 0.04% to 0.25% after 24-h culture (p < 0.001) and further elevation to 0.63% after 72-h chronic culture (p < 0.001). UCP2–KO islets had elevated basal insulin secretion from 0.05% to 0.4% after 24-h culture (p < 0.001) and further elevation to 0.80% after 72-h chronic culture (p < 0.001). Although after 24-h exposure to 25 mM high glucose condition, both WT and UCP2-KO islets maintained normal GSIS to a subsequent high glucose challenge, the UCP2-KO islets secreted significantly more insulin than that from WT islets (p < 0.001) (Fig. 3B). Interestingly, while the GSIS function of WT islets was significantly impaired after 72-h exposure to 25 mM high glucose condition (p > 0.1) (Fig. 3C), the GSIS function of UCP2-KO islets remained intact and maintained the ability to secrete insulin in response to a subsequent high glucose challenge (p < 0.005) (Fig. 3C). The insulin content was normalized with DNA content (Table 1).

UCP2-KO Islets Were Resistant to High Glucose and Proinflammatory Cytokine-Induced Death

To explore the underlying mechanism of UCP2 deficiency to mediate improvement of islet graft function, we studied the effect of UCP2 on islet viability and function in response to an adverse environment of hyperglycemia and proinflammation, which mimics the posttransplantation circumstance. We incubated freshly isolated islets (n = 50) for 24 h in the absence or presence of 25 mM high glucose and a mixture of IL-1β, IFN-γ, and TNF-α at the concentrations typically applied in previously published studies (21). Before confocal imaging analysis, double nuclear staining was applied using PI and Hoechst 33342 (for staining all the nuclei). As Figure 4 shows, approximately 50% of the islets from wild-type donors were PI staining positive, indicating they were nonviable. In contrast, only 28.1 ± 1.6% UCP2-KO islets were PI staining positive (p < 0.001), suggesting that the UCP2-deficient islets were resistant to high glucose and proinflammatory cytokine-induced death.

Figure 4.

UCP2 deficiency preserves the islets viability after acute high glucose challenge and cytokines stimulation. (A) Isolated islets from UCP2-KO or WT were challenged with 25 mM high glucose and cytokine cocktail for 24 h. Confocal imaging of islets stained with propidium iodide (labeling the dead cells in red fluorescence) and Hoechst 33342 (labeling all the cell nuclei in blue fluorescence). (B) Percent of cell death was calculated by dividing the number of dead cells after treatment divided by the total number of cells tested per treatment. At least 15 islets were counted for each group in each independent set of experiments. (C) Bcl-2 mRNA expression levels in islets cultured for 72 h at 25 mM high glucose were measured. Expression levels are expressed as relative to the WT group. White bar: group UCP2-WT; black bar: group UCP2-KO. Results are expressed as mean ± SEM from three independent repeats with six replicates. Bcl-2 gene relative to an arbitrary value of 1, which was assigned to the expression levels in WT. ^**p < 0.001 versus WT, unpaired, two-tailed Student's t-test. Scale bar: 100 μM.

To further explore the mechanism, we determined the mRNA expression levels of Bcl-2, a gene associated with cell survival (5), from UCP2-KO and WT islets, consistent with a previous report that the Bcl-2 is expressed at low levels from freshly isolated WT islets (20). However, after exposure to high glucose culture, UCP2-KO islets expressed twofold higher Bcl-2 mRNA levels in comparison with that from WT islets, which correlated with the resistance of UCP2-KO islets to the deleterious effects of high glucose challenge.

Discussion

β-Cells sense glucose through its metabolism, resulting in increasing ATP, which closes K_ATP channels leading to a depolarization of the cell membrane potential, increases Ca²⁺ influx, and finally stimulation of insulin secretion. UCP2 mediates mitochondrial proton leak, decreasing ATP production, and decreasing ATP/ADP ratio. Our previous study established that UCP2 negatively regulates GSIS (20). In the present study, we sought to examine the effects of UCP2 in the contest of syngeneic islet transplantation.

Studies have showed that an adequate mass of islets is critical to achieve primary islet graft function both in human and animal models (12). In our experimental setting, 300 islets isolated from both WT and UCP2-KO C57BL/6 donors were capable to reverse hyperglycemia in 3 days when transplanted into syngeneic streptozotocin-induced diabetic recipients (Fig. 1A). To examine the potency of UCP2-KO islets, we transplanted a suboptimal mass of 200 islets from either WT or UCP2-KO donors. When 200 islets from WT donors were transplanted into syngeneic diabetic recipients, they were unable to reverse hyperglycemia until up to 22 days posttransplantation (Fig. 1). Our study corroborates a recent reported that hyperglycemia correlated with the likelihood of primary nonfunction of marginal islet mass transplantation in rodent models (12,14). In contrast, 200 islets from UCP2-KO donors were able to rapidly normalize glucose levels in 1 week in all diabetic syngeneic WT recipients (Fig. 1B). These data indicated that UCP2-KO islets were more potent in reversing hyperglycemia in syngeneic diabetic recipients. The results of glucose tolerance test conducted on posttransplantation day 12 supported this conclusion. As elucidated in Figure 1B, the recipients receiving UCP2-KO islets exhibited normal glucose profiles following intraperitoneal infusion of glucose. The blood glucose levels were elevated to peak levels followed by a decline to prechallenge levels within 2 h (Fig. 1C). In contrast, the recipients receiving WT islets demonstrated an impaired pattern of GTT; the elevated glucose levels following glucose load decline slowly and remained at 245.0 ± 25.1 mg/dl upon 2 h post-glucose challenge (Fig. 1C).

In transplanted islets, the deleterious effects of hyperglycemia on β-cell function have been described in mice and humans (8,10,14). Impairment of GSIS plays an important pathophysiological role in the development of β-cell dysfunction. Indeed, our results showed that islets cultured in high glucose medium were associated with markedly increased UCP2 mRNA expression levels (Fig. 2). Moreover, the increased UCP2 mRNA expression in WT islets correlated with severely impaired GSIS and lost glucose sensing after chronic high glucose challenge (Fig. 3B, C). In contrast, UCP2-deficient islets enhanced the islet GSIS and maintained the ability to increase insulin secretion followed by acute and chronic hyperglycemic challenges (Fig. 3). These results coincide with the recent report of Krauss et al. that hyperglycemia caused activation of UCP2, and consequently impaired GSIS (11). Preserved GSIS in UCP2-KO islets in chronic hyperglycemic environment may confer a significant beneficial effect on primary function of islet grafts and contribute to the reversion of hyperglycemia in diabetic recipients.

It has been reported that more than half of the islets were lost in the initial days after syngeneic transplantation; and both apoptosis and necrosis contributed to this early β-cell death (2,3). Preserving islets after transplant is complex issues involving many factors, including mechanical damage induce by isolation process, hypoxia of transplanted islets, and the inflammatory and metabolic condition of the recipients. To further explore the mechanism by which marginal UCP2-KO islets restore normal glucose levels in diabetic recipients, we studied the effect of UCP2 on islet survival. As shown in Figure 4, after exposure of WT islets in high glucose and proinflammatory cytokine medium for 24 h approximately 50% WT islets were PI staining positive, indicating they lost viability. In contrast, only 28.1 ± 1.6% UCP2-KO islets were PI staining positive (p < 0.001), suggesting that the UCP2-KO islets were resistant to death induced by high glucose and proinflammatory cytokines (Fig. 4B). This observation coincides with the result that Bcl-2, a prosurvival factor, gene expression was about twofold higher in UCP2-KO islets than that of WT islets after hyperglycemia culture. Indeed, studies reported that overexpression of Bcl-2 in β-cell lines or islet partially protects them from cytokine-induced β-cell apoptosis (16).

In summary, our study explored a potential mechanism that may explain the benefit of UCP2-KO islets in islet transplantation. Hyperglycemia induced UCP2 expression, which uncouples the mitochondrial oxidative phosphorylation system, decreases the efficiency of energy synthesis, lowers ATP levels, and impairs islet GSIS and glucose sensing. Primary function was compromised when marginal islets were transplanted to diabetic recipients. In contrast, UCP2-KO islets preserved glucose sensing, GSIS, and viability after chronic hyperglycemia, thus ensuring the primary function of marginal islet grafts. Targeting UCP2 may provide a novel strategy to improve function of transplanted islets and reduce the number of islets required for transplantation. A small molecule, genipin, was shown recently to inhibit UCP2-mediated proton leak. In isolated mitochondria, genipin inhibits UCP2-mediated proton leak. In pancreatic islet cells, genipin increases mitochondrial membrane potential, increases ATP levels, closes KATP channels, and stimulates insulin secretion. It may provide an opportunity for this small molecule to target UCP2 and improve islets function (23).