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Original Articles: Cell Therapy and Islet Transplantation

Inducible Nitric Oxide Synthase-Nitric Oxide Plays an Important Role in Acute and Severe Hypoxic Injury to Pancreatic Beta Cells

Ko, Seung-Hyun1; Ryu, Gyeong Ryul1; Kim, SeungBum1; Ahn, Yu-Bae1; Yoon, Kun-Ho1; Kaneto, Hideaki2; Ha, Hunjoo3; Kim, Yu Seun4; Song, Ki-Ho1,5

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doi: 10.1097/TP.0b013e31816168f9
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Islet transplantation is a potential strategy for curing type 1 diabetes mellitus (1). Pancreatic beta cell failure is a fundamental pathogenic mechanism of both type 1 and type 2 diabetes mellitus (2–4). Because a reduced beta cell mass has been confirmed in type 2 diabetes in previous studies (5, 6), beta-cell replacement might also be a therapeutic alternative for type 2 diabetes mellitus.

Despite recent progress in islet transplantation (7), the requirement for an abundant islet mass is a major limitation to its feasible applicability. Moreover, the processes of islet isolation and purification can jeopardize graft survival because the islets are no longer attached to a vascular system until revascularization takes places. During this early posttransplantation period, nutrient deprivation and hypoxia are important determinants of graft survival and function (8). In addition to this hypoxic injury, there are several mediators that have been known to cause pancreatic islet dysfunction or cell death, including inflammatory cytokines such as interleukin-1β, tumor necrosis factor-α, and interferon-γ (9, 10).

Hypoxia activates a number of genes that are important in the cellular adaptation to low oxygen condition. These genes are transcriptionally regulated by hypoxia-inducible factor 1 (HIF-1), a heterodimeric basic helix-loop-helix transcription factor consisting of the two subunits HIF-1α and HIF-1β. While HIF-1β is constitutively expressed, hypoxia dependent HIF-1 induction occurs via the stabilization of the α subunit (11–13). The presence of HIF-1α has been clearly demonstrated in the beta cell line, isolated rat and human islets when exposed to low oxygen concentrations (14). Recently, Miao et al. reported that early transplanted islets strongly express HIF-1α in association with beta cell death until adequate revascularization is established (15). Most target genes of HIF-1 are involved in energy metabolism, iron homeostasis, angiogenesis, and cell proliferation or survival. Inducible nitric oxide synthase (iNOS) is possibly also induced by HIF-1 (16, 17). Thus, it is possible that hypoxic stress facilitates cell protection or injury by modulating iNOS expression in beta cells.

Nitric oxide (NO) is a short-lived free radical that plays an important role as a regulator of diverse pathophysiological mechanisms in the cardiovascular, nervous and immunological systems. Among at least three isoforms of NOS, iNOS is induced by cytokines or oxidative stress under inflammatory conditions (16, 17). It is well known that NO, generated by iNOS, is a potential mediator of cytokine-induced beta cell dysfunction (18). NO causes cell death by inducing DNA strand breaks, leading to apoptosis in various cell types (19–21). However, the exact role of iNOS-NO remains obscure in hypoxic injury to beta cells.

In this study, we investigated whether acute and severe hypoxic injury affects iNOS-NO signaling in primary rat islets and the rat beta cell line (INS-1).


Cell Culture

Primary rat islets were isolated from 8-week-old male Sprague Dawley rats (∼200 g) using collagenase P (Roche, Mannheim, Germany) and subsequent separation on Ficoll gradient (Biochrom AG, Berlin, Germany) as previously described (22). The islets and INS-1 cells (kindly provided by Dr. Kyu-Chang Won, Yeungnam University, Korea) were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium at 37°C in 95% air and 5% CO2.

To induce acute and severe hypoxic conditions, the islets (200 islets/35 mm dish with 3 ml of the culture medium) and INS-1 cells (5×105 cells/well in a 12-well plate) were preincubated with the aforementioned medium for 16 hr, and then incubated in an anoxic chamber (95% N2, 5% CO2) at 37°C for up to 12 hr. The partial oxygen pressure (PO2) of the culture medium, as measured by a blood gas analyzer, was approximately 10 mmHg.

For some experiments, 100 μM 1400W (N-(3-[amino-methyl)benzyl] acetamidine) (Sigma, St. Louis, MO), a specific iNOS inhibitor (23), was added to the medium 2 hr prior to hypoxic exposure.

Cell Viability Analysis

Cell viability was determined with acridine orange (AO)/propidium iodide (PI) staining or Cell Counting Kit 8 (Dojindo Laboratories, Kumamoto, Japan) using 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-8). For AO/PI staining, a minimum of 500 cells were counted in at least three random fields for each experiment. The percentage of dead cells (cell death rate) was determined by counting the total number of PI-positive cells divided by the total number of PI and AO-positive cells. Cell Counting Kit 8 is based on the ability of viable cells to reduce tetrazolium salt to soluble colored formazan. Cells (5×105/well for INS-1 cells and 200 islets/well for rat islets) were seeded in 12-well plates on the day before the experiment. After the medium was removed, 10 μl of CCK-8 solution was added to each well of the plate. The plate was incubated for 4 hr. The absorbance was measured at 450 nm using a microplate reader.

Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick-End Labeling (TUNEL) Assay

The TUNEL assay was performed to detect apoptosis in rat islets. Rat islets were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), pelleted in 2% agar, and embedded in paraffin. The tissue sections were deparaffinized and rehydrated through three changes of xylene and graded alcohol, washed in PBS for 5 min, and then incubated in 20 μg/ml proteinase K for 15 min at room temperature. The ApopTag Fluorescein In Situ Apoptosis Detection Kit (Chemicon International, Temecula, CA) was used according to manufacturer's instructions. Briefly, endogenous peroxidase activity in the sections was blocked by incubation for 5 min with 3% H2O2 in PBS, followed by incubation with equilibration buffer. The sections were then incubated for 60 min at 37°C with terminal deoxynucleotidyl transferase enzyme in reaction buffer. The reaction was terminated by incubation with stop buffer at room temperature. Apoptotic cells were identified by fluorescein isothiocyanate (FITC) staining. Nuclei were stained with 4′,6′-diamidino-2-phenylindole (DAPI).

iNOS Immunostaining

The INS-1 cells and rat islets were fixed with 4% paraformaldehyde in PBS and stained with rabbit polyclonal antibody directed against rabbit iNOS (1:200; BD Transduction Laboratories, Palo Alto, CA) as the primary antibody and antirabbit Texas red (Jackson ImmunoResearch, West Grove, PA) as the secondary antibody. Nuclei were stained with DAPI.

NO Production in Culture Medium

NO production was measured in the culture medium as nitrite accumulation using the Griess reagent method (Promega, Madison, WI). NO data were expressed as nitrite concentrations per 200 rat islets or per DNA content (microgram) of INS-1 cells. INS-1 cells were treated with 100 ng/l interleukin-1β (Calbiochem, San Diego, CA) for 24 hr as the positive control. All samples were measured in triplicates.

Reverse Transcription and Quantitative Polymerase Chain Reaction (PCR)

Total RNA from cells cultured under each set of conditions was extracted with TRIzol Reagent (Invitrogen, Grand Island, NY, USA). Total RNA (2 μg) was used to synthesize first-strand cDNA according to the protocol of the SuperScript Preamplification System (Invitrogen). Total RNA was incubated with 0.5 μg oligo(dT)primer at 85°C for 3 min, and the reaction was then carried out in a mixture of 5 × first-strand buffer from the kit containing 50 mM Tris-HCl, 75 mM KCl, and 3 mM MgCl2, and 10 mM dithiothreitol, and 0.5 mM deoxynucleotide phosphates in a final volume of 25 μL. The contents of the tubes were incubated at 25°C for 10 min, Superscript II was added, and the reactions incubated at 42°C for 60 min. After the reaction, the enzyme was inactivated at 70°C for 15 min. The final solution was used directly for PCR amplification. The relative expression levels of genes were determined with reference to the expression of the cyclophilin gene. The PCR primers were: iNOS (689 bp), sense 5′-GGTCCAACCTGCAGGTCTT-3′, antisense 5′-GGTCCATGATGGTCACATT-3′; and cyclophilin (400 bp), sense 5′-AACCCCACCGTGTTCTTC-3′, antisense 5′-TGCCTTCTTTCACCTTCCC-3′. The PCR products were visualized by 1.5% agarose gel electrophoresis, and the density of each band was measured with a densitometry (Amersham Pharmacia Biotech, Buckinghamshire, UK).

Western Blot Analysis

For the western blot analysis, INS-1 cells and islets were collected at various time points during hypoxic injury, and then homogenized on ice in lysis buffer (150 mM NaCl, 50 mM Tris-HCl [pH 7.4], 2 mM ethylenediamine tetraacetic acid, 1% NP-40, 10 mM NaF, 1 mM Na3VO4, 10 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 0.1 mg/ml soybean inhibitor). The lysate was centrifuged at 15,000 rpm for 5 min at 4°C. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane (Hybond-P, Amersham Pharmacia Biotech). The membrane was blocked with 5% fat-free dry milk for 1 hr in Tris-buffered saline (pH 7.6) and then incubated overnight at 4°C with the primary antibodies. The antibodies used for immunoblotting were as follows: anti-iNOS antibody (1:1000; BD Transduction Laboratories), antiactivated caspase-3 antibody (1:2000; BD Transduction Laboratories), anti-HIF-1α (1:1000; Santa Cruz Biotechnology Inc., Santa Cruz, CA), antiphosphorylated c-Jun N-terminal kinase (JNK) 1/2, antitotal JNK1/2, antiphosphorylated extracellular signal regulated kinase (ERK) 1/2, antitotal ERK1/2, antiphosphorylated p38, and antitotal p38 (Cell Signaling Technology Inc., Beverly, MA). After the membranes were washed, they were incubated with secondary peroxidase-conjugated antirabbit or antimouse antibodies (Amersham Pharmacia Biotech) diluted 1:2000 in Tris-buffered saline with 0.01% Tween 20. Antibody detection system (ECL, Amersham Pharmacia Biotech) was used and the membranes were exposed to x-ray film. Protein band intensities were quantified with a VDS densitometer (Amersham Pharmacia Biotech).

Statistical Analysis

Data are expressed as means±SEM. Differences between groups were evaluated using the SPSS 10.0 program (SPSS Inc., Chicago, IL). The independent t test and one-way analysis of variance were used to analyze the quantitative variables between groups. A P value of < 0.05 was deemed significant.


Hypoxic Injury to INS-1 Cells and Isolated Islets

After incubation in the anoxic chamber, marked cell death occurred rapidly in a time dependent manner in both rat islets and INS-1 cells (Fig. 1A, B). To examine whether apoptosis is induced by hypoxic injury in INS-1 cells and rat islets, we performed western blot analysis with antiactivated caspase-3 antibody. As shown in Figure 1C, within 2 hr of exposure to hypoxia, increased levels of activated caspase-3 were detected in both INS-1 cells and isolated islets, suggesting that apoptosis was induced in the process of cell death. Using TUNEL assay, we observed that apoptotic cells were significantly increased after hypoxia in islets (percentage of TUNEL-positive cells: 1.6±0.1% at 0 hr vs. 5.1±0.1% at 4 hr, n=3, P<0.05; Fig. 1D).

Cell viability, caspase-3 activation, and TUNEL assay after hypoxic exposure. (A) Bar graphs show that cell death rate (percentage of PI-positive cells) increased with time in both INS-1 cells and rat islets during hypoxia; n=3. *P<0.05 vs. 0 hr. (B) Representative examples of AO (green)/PI (red) staining in INS-1 cells 0 hr and 6 hr after hypoxia. Magnification, ×400. (C) Western blot analysis, representative of 3 individual experiments, showed that the expression of activated caspase-3 in both INS-1 cells and rat islets increased with time during hypoxia. (D) TUNEL assay, representative of three individual experiments under a fluorescent microscope, showed increased apoptotic cells (FITC-positive) in rat islets 4 hr after hypoxia. Nuclei were stained with DAPI (blue). Magnification, ×400.

Effect of Hypoxia on HIF-1α Expression, NO Production, and iNOS Expression

Because HIF-1α is a central factor in the cellular response to hypoxic conditions in various cell types, we examined whether HIF-1α expression is induced by hypoxic injury in INS-1 cells. As shown in Figure 2A, hypoxia induced an increase in HIF-1α protein expression in INS-1 cells. The increase was seen as early as 1 hr after exposure to hypoxia and was maintained for 4 hr.

Induction of HIF-1α, nitrite production, and iNOS mRNA expression after hypoxic exposure. (A) Western blot analysis showed that expression of HIF-1α in INS-1 cells increased with time during hypoxia. Bar graphs show the quantified expression of HIF-1α after normalization to that of β-actin, expressed as fold changes from 0 h; n=3. (B) Nitrite accumulation in the culture medium of both INS-1 cells and rat islets increased significantly with time during hypoxia; n=3. (C) Reverse-transcription PCR showed that iNOS mRNA expression increased with time in both INS-1 cells and rat islets during hypoxia. Bar graphs show the quantified expression of iNOS after normalization to that of cyclophilin, expressed as fold changes from 0 h; n=3. *P<0.05 vs. 0 hr.

We examined NO production by measuring nitrite accumulation over the hypoxic period. As shown in Figure 2B, the nitrite concentration in the culture medium increased in a time-dependent manner in both INS-1 cells and islets.

Next, to examine whether NO production is induced by iNOS expression, we estimated iNOS mRNA expression after hypoxic injury by reverse-transcription PCR analysis. As shown in Figure 2C, iNOS mRNA was expressed increasingly during hypoxia in both INS-1 cells and islets. iNOS protein expression determined by western blotting also increased significantly in INS-1 cells 2 hr and 4 hr after hypoxia (Fig. 3A). Immunostaining for iNOS in INS-1 cells or rat islets exposed to hypoxia for 4 hr confirmed the presence of iNOS protein in the cytoplasm of the cells (Fig. 3B).

iNOS protein expression after hypoxic exposure. (A) Western blot analysis showed that the expression of iNOS protein in INS-1 cells increased significantly with time during hypoxia. Bar graphs show the quantified expression of iNOS after normalization to that of β-tubulin, expressed as fold changes from 0 h; n=3. *P<0.05 vs. 0 hr. (B) Immunostaining confirmed the presence of iNOS protein (red) in the cytoplasm of INS-1 cells and rat islets 4 hr after hypoxia. Nuclei were stained with DAPI (blue). Magnification, ×400.

Mitogen-Activated Protein Kinase (MAPK) Activation After Hypoxic Injury

To determine which kinase is activated and possibly involved in the induction of apoptosis, we examined the changes in various kinases after hypoxic injury. As shown in Figure 4A, hypoxia activated both the JNK and the ERK pathways, whereas the phosphorylation of p38 was not affected.

MAPK activation after hypoxic exposure. (A) Western blot analysis showed that the phosphorylation of ERK (p-ERK1/2) and JNK (p-JNK1/2) was increased with time in INS-1 cells during hypoxia, whereas p38 phosphorylation (p-p38) was not affected. (B, C) Bar graphs show the quantified expression of p-JNK1/2 after normalization to that of JNK1/2, expressed as fold changes from 0 hr. Pretreatment with 1400W partially inhibited hypoxia-induced JNK phosphorylation as well as expression of activated caspase-3. Western blotting data are representative of five individual experiments. *P<0.05 vs. 0 hr, **P<0.05 vs. 4 hr without 1400W.

Next, we examined the effect of iNOS inhibition on hypoxia-induced JNK phosphorylation. The addition of 1400W to INS-1 cells partially inhibited hypoxia-induced JNK phosphorylation (Fig. 4B) as well as expression of activated caspase-3 (Fig. 4C), though there was no significant effect of 1400W at 0 and 2 hr of hypoxia (data not shown).

Moreover, using Cell Counting Kit 8, we observed that 1400W pretreatment significantly prevented cell death in both INS-1 cells and islets (Fig. 5).

Effect of 1400W pretreatment on cell viability after hypoxic exposure. 1400W pretreatment significantly attenuated decrease in cell viability, as measured by Cell Counting Kit 8, in both INS-1 cells and rat islets 4 hr after hypoxia. n=3. *P<0.05 vs. 0 hr, **P<0.05 vs. 4 hr without 1400W.

We also found that treatment with 500 μM deta-NO, an NO donor, for up to 180 min in INS-1 cells under normoxic conditions induced the phosphorylation of JNK2, ERK2, and p38, suggesting that NO activates MAPKs directly (data not shown). Taking these data together, we assume that NO is, at least in part, involved in the activation of the JNK pathway after hypoxic injury.


In this study, we demonstrated that acute and severe hypoxic injury can activate iNOS-NO signaling together with JNK phosphorylation and HIF-1α expression in pancreatic beta cells and that pretreatment with a specific iNOS inhibitor attenuated beta cell death.

Islet transplantation is a potential strategy for curing type 1 diabetes mellitus (1, 7). Recently, the outcomes of islet transplantation have been improved dramatically. However, a number of problems remain, such as chemical and mechanical injury to the islets during the isolation procedure, immunosuppressive agents, and limited islet sources (24).

During the isolation process, the islets are separated from their blood supply. Perfusion of the transplanted islets does not occur until after the process of angiogenesis has established new capillaries of donor origin. This delayed arterial perfusion leads to temporary hypoxic injury to the islets (25, 26). As expected, hypoxia can cause pancreatic islet death and dysfunction (27). Thus, it might be true that the outcome of islet transplantation is in part dependent on sufficient oxygen delivery to the islets.

Under low oxygen conditions, a multitude of genes that are essential for cellular responsiveness and survival are the immediate early targets of various transcription factors (12). In particular, HIF-1 is a central effector of the cellular response to hypoxic conditions (28). HIF-1 is expressed in pancreatic beta cell lines and isolated rat and human islets when exposed to hypoxia (14). The present study also demonstrated that hypoxia induced an increase in HIF-1α expression in INS-1 cells within 1 hr. The O2-mediated regulation of iNOS expression is so complex that, depending on the cell type, hypoxia can increase, decrease, or not affect cellular iNOS levels (17, 29–31).

iNOS-NO has been implicated in apoptotic cell death (19–21). In pancreatic beta cells, the direct deleterious effects of iNOS-NO include the inhibition of insulin secretion (32), mitochondrial dysfunction, DNA damage, and the production of reactive oxygen species (ROS) (33, 34). However, the role of iNOS-NO signaling in hypoxic injury to pancreatic beta cells has not yet been studied in detail. In this study, we observed that iNOS expression, NO production, and HIF-1α were induced by hypoxic injury in INS-1 cells and rat islets. Hypoxia caused marked cell death with caspase-3 activation, but 1400W, a specific inhibitor of iNOS (23), improved cell viability in INS-1 cells.

Hypoxia can induce signals into all the major MAPK cascades, including ERK, JNK, and p38 as well as JAK/STAT (signal transducer and activator of transcription) pathways (12, 35). Among these, we found that the phosphorylation of JNK was significantly increased in response to hypoxic cell damage, and that iNOS inhibition partially inhibited hypoxia-induced JNK phosphorylation. Moreover, treatment with deta-NO, an NO donor, activated MAPKs including JNK, as reported previously (19). Considering these data together, we assume that iNOS-NO is, at least in part, involved in the activation of the JNK pathway after hypoxic injury. In fact, JNK in the pancreas is activated during brain death, pancreas procurement, and organ preservation, and its activity increases progressively during the isolation procedure (36). Very recently, JNK inhibition was reported to confer protection against apoptosis induced during islet preparation (37) and islet graft loss after transplantation (38).

Another possible mechanism underlying hypoxia-mediated beta cell death could act via oxidative stress because hypoxia is known to increase ROS (39). Oxidative stress and the consequent activation of the JNK pathway are thought to be important in the progression of the beta cell dysfunction found in type 2 diabetes mellitus (40). However, further studies are required to investigate whether hypoxia increases ROS production in beta cells.

The limitations of this study include the fact that we did not address the functional consequences of iNOS-NO activation under hypoxic conditions in beta cells. Interestingly, Dionne et al. (27) reported that a 50% decrease in the second phase of insulin secretion was observed in perifused islets when PO2 was lowered to 10 mmHg in the perifusate. Second, beta cell death under the experimental conditions could be also affected by proinflammatory cytokine expression from the islets, although there was a clear association between hypoxia and activation of HIF-1α, iNOS, JNK, and caspase-3.

In conclusion, we have demonstrated that iNOS-NO plays an important role in acute and severe hypoxic injury to pancreatic beta cells. Therefore, iNOS-NO might be a potential therapeutic target for ensuring beta cell survival in islet transplantation through the prevention of cell death from hypoxia.


We thank Min-Kyung Lee for her technical assistance.


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Hypoxia; Islet transplantation; Inducible nitric oxide synthase; Nitric oxide; c-Jun N-terminal kinase

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