• apoptosis;
  • Bax;
  • Bcl-2;
  • β-cells;
  • calbindin-D28k;
  • insulin;
  • intracellular calcium;
  • TNF-α


  1. Top of page
  2. Abstract
  6. Acknowledgements

The migration of macrophages and lymphocytes that produce cytokines such as tumor necrosis factor-α (TNF-α) causes β-cell death, leading to type 1 diabetes. Similarly, in type 2 diabetes, the adipocyte-derived cytokines including TNF-α are elevated in the circulation, causing inflammation and insulin resistance. Thus, the studies described in this article using TNF-α are relevant to furthering our understanding of the pathogenesis of diabetes mellitus. We used RINr1046-38 (RIN) insulin-producing β-cells, which constitutively express calbindin-D28k, to characterize the effect of TNF-α on apoptosis, replication, insulin release, and gene and protein expression. Western blots of TNF-α-treated RIN cells revealed a decrease in calbindin-D28k. By ELISA, TNF-α-treated β-cells had 47% less calbindin-D28k than controls. In association with the decline in calbindin-D28k, TNF-α treatment of RIN cells led to a 73% greater increase in changes in intracellular calcium concentration (Δ[Ca2+]i) in TNF-α-treated cells as compared to that in control RIN cells upon treatment with 50 mM KCl; caused a greater increase in the [Ca2+]i following the addition of 5.5 μM ionomycin; increased by more than threefold the apoptotic rate, expressed as the percentage of TUNEL-positive nuclei to total nuclei; decreased the rate of cell replication by 36%; and increased and decreased selectively the expression of specific genes as determined by microarray analysis. The subcellular localizations of Bcl-2, an antiapoptotic protein, and Bax, a proapoptotic protein, within RIN cells were altered with TNF-α treatment such that the two were colocalized with mitochondria in the perinuclear region. We conclude that the proapoptotic action of TNF-α on β-cells is manifested via decreased expression of calbindin-D28k and is mediated at least in part by [Ca2+]i. © 2005 Wiley-Liss, Inc.

Cytokines are small secreted proteins that act as signaling molecules, much like hormones (Wajant et al., 2003). Tumor necrosis factor-α (TNF-α), a soluble cytokine and neurotropic factor that is produced upon activation by the immune system, has diverse actions on cells. TNF-α is known to enhance resistance of neurons and normal nonneuronal cells to Ca2+ cytotoxicity, oxidative stress, and acidosis, yet TNF-α leads to apoptosis in many types of tumor cells, including mouse insulinoma (MIN6) cells as well as tumor necrosis in certain animal model systems (Cheng et al., 1994; Barger et al., 1995; Mattson et al., 1997a; Ishizuka, et al., 1999). Two spontaneous rodent models of insulin-dependent diabetes mellitus (IDDM; type 1), the BioBreed (BB) rat and nonobese diabetic (NOD) mouse, are protected from diabetes by TNF-α (Satoh et al., 1989, Satoh et al., 1990; Jacob et al., 1990; Flavell et al., 1996; Grewal et al., 1996). However, the migration of macrophages and lymphocytes that produce cytokines such as TNF-α to the pancreatic islets causes destruction of β-cells, leading to IDDM (Rabinovitch et al., 2001). Additionally, in type 2 diabetes, the adipocyte-derived cytokines such as TNF-α are elevated in the blood, causing an inflammatory response and insulin resistance (Donath et al., 2003). It has been proposed that expression of calbindin-D28k can protect pancreatic islet β-cells from autoimmune destruction in type 1 diabetes (Rabinovitch et al., 2001). In neurons, TNF-α is known to exert cytoprotective effects by inducing the expression of genes important in controlling intracellular Ca2+ concentration, [Ca2+]i, including calbindin-D28k (Mattson et al., 1995, Mattson et al., 1997a, Mattson et al., 1997b; Bruce et al., 1996; Eizirik et al., 1996).

Wang et al. (1999) have shown that cytokines can activate a low voltage-dependent Ca2+ current in mouse βTC-3 cells and also increase the [Ca2+]i in mouse islet cells, leading to DNA fragmentation and cell death. Intracellular Ca2+ regulates many key steps in signal transduction pathways from initiating early signaling events to causing irreversible changes leading not only to apoptosis, but to proliferation and differentiation as well (Mattson, 1992; McConkey and Orrenius, 1996).

Expression of Bcl-2 can protect cells from both calcium-induced and oxidative stress injury (Marin et al., 1996; Xie et al., 1996; Rabinovitch et al., 1999). Bcl-2 localizes in mitochondria and it inhibits mitochondrial release of cytochrome c that can trigger apoptosis via activation of caspases as well as generation of the superoxide radical; therefore, Bcl-2 protects β-cells from apoptosis (Rabinovitch et al., 1999). Proapoptotic members of the Bcl-2 family of proteins such as Bax and antiapoptotic protein Bcl-xL reside mainly in the cytoplasm, but can translocate to mitochondria and promote cytochrome c release in response to various apoptotic stimuli (Shimizu et al., 1999; Saito et al., 2000).

Calbindin-D28k is a predominantly cytosolic high-affinity calcium-binding protein that is found in many tissues, including kidney, brain, and pancreatic islets (Christakos et al., 1989). Luo and Welsh (1997) reported that cytokine-induced apoptosis was partially blocked by calbindin-D28k. This antiapoptotic effect of calbindin-D28k has been attributed to alterations of Ca2+ signaling pathways and inhibition of caspase-3 activity (Bellido, et al., 2000). Hence, it is important to assess further the role of TNF-α in β-cells, both in terms of regulation of gene expression of calbindin-D28k and the effect of TNF-α on β-cell function, as reflected in insulin secretion and Ca2+ regulatory mechanisms. We report here the effects of TNF-α on apoptosis, cell division, [Ca2+]i, insulin secretion, calbindin-D28k, localization of Bcl-2, and Bax, as well as gene transcription on RINr1046-38 (RIN) insulin-producing β-cells.


  1. Top of page
  2. Abstract
  6. Acknowledgements

Cell Culture and Protocol

RINr1046-38 insulin-producing β-cells, kindly provided by Dr. Bruce Chertow (Veterans Administration Medical Center, Huntington, WV), were grown on 25 mm diameter glass coverslips in six-well culture plates in a 5% CO2 incubator at 37°C. The starting cell count per well was ∼ 1 × 105. Cells were also grown in 75 cm2 culture flasks. The cell culture medium was RPMI-1640 supplemented with 10% (w/v) fetal bovine serum, 100 units/mL of penicillin, and 100 μg/mL of streptomycin (Gibco-BRL, Gaithersburg, MD). After 5 days of culture, RIN cells were exposed to 24-hr treatment with 10 ng/mL TNF-α (rat, recombinant; Chemicon International, Temecula, CA). For replication studies, after 16–18 hr of this incubation period, 5-bromo-2′-deoxyuridine (5-BrdU; Sigma-Aldrich, St. Louis, MO) was added to each well to a give a final concentration of 10 μg 5-BrdU/mL of cell culture medium. The cells were further incubated for 6 hr.

Assessment of Apoptosis by TUNEL Method

TNF-α-mediated rates of apoptosis of individual β-cells were assessed in situ by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method for detection of DNA fragmentation. Rates of apoptosis were determined on β-cells grown on coverslips as described above, fixed in 4% (w/v) freshly made paraformaldehyde in 0.1 M PBS, pH 7.2, for 1 hr at room temperature, and processed by routine methods. Cells were treated with proteinase K (20 μg/ml in 10 mM Tris, pH 7.6, 30 min at 37°C) and incubated with terminal deoxynucleotidyl transferase (0.5 U/μl) and fluoresceinated dUTP (50 μM; Boehringer Mannheim, Mannheim, Germany) for 1 hr at 37°C in the dark. Staining with Hoechst 33258 (0.5 μg/ml) was used to visualize nuclei. The incorporated fluorescein-dUTP (green) and Hoechst (blue) images were acquired on a Microphot microscope (Nikon USA, Melville, NY) and captured and stored using a 24-bit SPOT-RT digital color camera and software (Diagnostic Instruments, Sterling Heights, MI). Apoptotic rate was expressed as the number of FITC-positive (apoptotic) nuclei per total number of β-cells nuclei counted. At least 250 nuclei from three cultures of each group were counted.

Measurement of Replication With 5-BrdU

Replication was monitored in situ with 5-BrdU incorporation and staining of nuclei. 5-BrdU is a thymidine analogue, which is incorporated into DNA during the S-phase of the cell cycle. 5-BrdU (10 μg/mL; Sigma) was added to media 16–18 hr following the addition of TNF-α. After 6 hr, the duration of G2 + M phases of rat β-cells (Swenne, 1982), the incorporation of 5-BrdU into cells was stopped by adding freshly prepared Bouin's solution. The cells incorporating 5-BrdU were then determined using fluorescent-labeled Alexa Fluor 594-conjugated anti-BrdU IgG (Molecular Probes, Eugene, OR). The total number of nuclei was determined using Hoechst 33258 fluorescent nuclear dye. Fluorescence images were obtained as described above. Replication rate was expressed as the number of 5-BrdU-positive nuclei per total number of β-cell nuclei counted. At least 250 nuclei from at least five cultures of each group were counted.

Western Analysis of Proteins

Levels of calbindin-D28k, β-actin as a control protein, and apoptosis-regulating proteins Bcl-2, Bax, and Bcl-xL were evaluated by Western blotting in TNF-α-treated and control β-cells. Cell lysates at 50 μg total protein per well were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes (Schleicher and Schuell) in a Bio-Rad Mini-Protein apparatus. Membranes were blocked for 1 hr at room temperature in blocking buffer. A mouse monoclonal anticalbindin-D28k (clone CL-300; Sigma) was used for calbindin-D28k. To evaluate levels of the anticell death proteins Bcl-2 and Bcl-xL, we used rabbit polyclonal antibodies with broad species cross-reactivity (Santa Cruz). Cross-reacting proteins were visualized with alkaline phosphatase-conjugated secondary antibodies and 5-bromo-4-chloro-3-indolylphosphate p-toluidine/nitroblue tetrazolium chloride (BCIP/NBT).

ELISA for Calbindin-D28k and Insulin

Levels of insulin secretion and calbindin-D28k were quantitated by enzyme-linked immunosorbent assay (ELISA) as previously described (Kekow et al., 1988; Sergeev and Rhoten, 1998; Parkash et al., 2002). The insulin assay was modified as follows, while the calbindin-D28k was determined as referenced above except that the coating protein, rat recombinant calbindin-D28k, was purchased from Swant (Belliuzona, Switzerland) and the wells were coated with 20 ng/well/150 μl of coating buffer. An affinity-purified antiguinea pig IgG raised in rabbit (Sigma-Aldrich) was used at 1:500 dilution of 1 mg/mL stock to coat the 96-well plate. The plate was then washed with ELX 50 (Bio-Tek Instruments) plate washer and incubated for 4 hr at 37°C with guinea pig anti-insulin (1:200 k diluted). The plate was washed and standards and samples (100 μL/well) along with peroxidase-labeled insulin were added to appropriate wells. The plate was incubated overnight at 40°C on a shaker. Then, 150 μL of substrate O-phenylenediamine dichloride solution was added to each well and the plate was incubated for 30 min at room temperature. The reaction was stopped with 50 μL of 1 M H2SO4/well and the plate was read in a μQuant (Bio-Tek Instruments) reader at 490 nm. Total protein concentrations were determined by Bio-Rad Dc microassay procedure as per the manufacturer's protocol.

RT-PCR for Gene Expression of Bcl-2 Family Members

Levels of gene expression of Bcl-2 and Bax were assessed by reverse transcriptase-polymerase chain reaction (RT-PCR). Total RNA was isolated from RIN cells using Trizol reagent (Invitrogen Life Technologies). Total RNA (500 ng/reaction) with a 260/280 ratio of 1.8 or higher was converted into single-stranded cDNA using an oligonucleotide primer (oligoDT; USB) and extension by an RNA-dependent DNA polymerase. cDNA was amplified by PCR using primers to Bcl-2 and Bax, as well as GAPDH (APO-PCR; Sigma). The Bax PCR primer set (product B8304; Sigma) contained the following sequence: reverse primer sequence (3′ antisense), 5′-CATCTTCTTCCAGATGGTGA-3′; forward primary sequence (5′ sense), 5′-GTTTCATCCAGGATCGAGCAG-3′. Similarly, the Bcl-2 PCR primer set obtained from Sigma (product B9179) contained reverse primer sequence (3′ antisense), 5′-GAGACAGCCAGGAGAAATCA-3′; forward primer sequence (5′ sense), 5′-CCTGTGGATGACTGAGTACC-3′. The control housekeeping gene GAPDH had its PCR primer set (product P7732; Sigma) as follows: reverse primer sequence (3′ antisense), 5′-YGCCTGCTTCACCACCTTC-3′; forward primer sequence (5′ sense), 5′-TGCMTCCTGCACCACCAACT-3′, where M = A or C and Y = T or C. The PCR program was run at 95°C for 2 min, 94°C for 45 sec/53°C for 45 sec/72°C for 1.5 min for 40 cycles, and 72°C for 7 min. PCR products were separated by 2% agarose gel electrophoresis and stained with ethidium bromide.

Microarray Analysis

The difference in gene expression profile between control (untreated) and TNF-α-treated RIN cells was carried out at the Marshall University DNA Core Facility. The PanChip 5.0 slides (Scearce et al., 2002) were obtained from the Vanderbilt Microarray Shared Resources and Functional Genomic Core, University of Pennsylvania. These particular slides contain 3,840 cDNA spots, including pancreas-specific genes, and positive and negative controls.

The total RNA from RIN cells was extracted by using the acid-phenol extraction procedure of Chomczynski and Sacchi (1987). Total RNA (40 μg) from both untreated and TNF-α-treated cells was used and labeled using Superscript II Reverse Transcriptase Labeling Kit (Invitrogen) and Cy3-dCTP and Cy5-dCTP (Perkins Elmer) according to the manufacturer's protocol. Following removal of unincorporated oligonucleotides, equal amounts of Cy3- and Cy5-labeled cDNA were mixed together and hybridized with PanChip 5.0 slides for 16 hr. Slides were then washed and dried and scanned using a ScanArray Express scanner (Perkin Elmer). The resultant images were analyzed using GeneSpring analytical software (Agilent).

Profiling was repeated six times with three biological and six technical repeats (three standard and three flip arrays). Only genes that changed their expression more than twofold (in either direction) in all six experiments were considered positive.

Ca2+ Regulatory Mechanisms and Insulin Secretion

[Ca2+]i was measured by using Fura-2 (Molecular Probes) ratio fluorescence microscopy as previously described (Parkash et al., 2004). The effect of calbindin-D28k content on Ca2+ regulatory mechanisms was determined by incubating β-cells in 0.1 mM carbachol, a cholinergic agonist that increases cytosolic Ca2+ through inositol trisphosphate signaling. Ionomycin, a nonfluorescent calcium ionophore, was used at 100 nM to 5.5 μM. The levels of insulin secreted by RIN cells into the incubation media were determined by ELISA as described above. To assess Ca2+-buffering capacity in β-cells following treatment with TNF-α, ionomycin was added to a final concentration of 5.5 μM to produce a nonphysiological rise in [Ca2+]i. The records of responses of individual β-cells were compared between TNF-α-treated and untreated β-cells. We also computed and compared the mean peak [Ca2+]i for TNF-α-treated and control cells.

Data Analysis

The data obtained on apoptosis, β-cell replication, insulin secretion, in situ quantification of calbindin-D28k, Bcl-2, Bax, Bcl-xL, and control proteins, Ca2+ regulation as reflected in the [Ca2+]i, intracellular distribution of calbindin-D28k, and microarrays were compared for control and TNF-α-treated β-cells. Statistical analysis of the data included Student's t-test, paired or unpaired, analysis of variance (ANOVA) as appropriate, and determination of correlation coefficients. The statistical analyses were carried out with applied statistical software (SigmaStat).


  1. Top of page
  2. Abstract
  6. Acknowledgements


The apoptotic rate in TNF-α-treated RIN cells appeared to be increased (Fig. 1). The ratio of TUNEL-positive nuclei to total nuclei (Hoechst-positive) was 0.051 ± 0.005 in TNF-α-treated RIN cells (n = 3) compared to 0.016 ± 0.002 in control cells (n = 4). The greater than threefold increase in TNF-α-treated cells apoptotic rate was highly significant (P < 0.001).

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Figure 1. TUNEL method to detect apoptosis in RIN cells. TNF-α-treated RIN cells (right) show more fragmented nuclei than control cells (left) as indicated by white arrows. Scale bar = 10 μm.

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Accompanying the increased apoptotic rate in TNF-α-treated RIN cells was a decrease in replication (Fig. 2). BrdU incorporation in TNF-α-treated cells was 0.138 ± 0.020 BrdU-positive nuclei/total Hoechst-positive nuclei (n = 5) compared to 0.215 ± 0.016 for control cells (n = 6; P = 0.014).

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Figure 2. BrdU-positive nuclei in RIN cells. RIN cells were incubated with 5-BrdU for 6 hr and cells incorporating BrdU were detected using anti-BrdU IgG conjugated to Alexa Fluor 594 (red). All nuclei were detected using Hoechst 33258 (blue). A: BrdU-positive nuclei in control RIN cells. BrdU-positive nuclei are seen as red with anti-BrdU IgG conjugated to Alexa Fluor 594 (top). Total nuclei are seen as blue with Hoechst 33258 staining (bottom). B: BrdU-positive nuclei in TNF-α-treated RIN cells. BrdU-positive nuclei are seen as red with anti-BrdU IgG conjugated to Alexa Fluor 594 (top). Total nuclei are seen as blue with Hoechst 33258 staining (bottom). BrdU-positive nuclei were reduced in the TNF-α-treated RIN cells. Scale bar = 8 μm.

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[Ca2+]i and Insulin Secretion

The physiological outcome of RIN cells cultured under the influence of TNF-α was evaluated by measuring [Ca2+]i and insulin secretion. There was a more pronounced increase in [Ca2+]i in response to 50 mM KCl in TNF-α-treated RIN cells (Fig. 3). Upon addition of 50 mM KCl, the [Ca2+]i increased to 1,362 ± 123 nM in control RIN cells (n = 28 cells) and to 2,223 ± 202 nM in TNF-α-treated RIN cells (n = 20 cells). The mean basal [Ca2+]i determined in HBSS was 150 ± 9 nM in control RIN cells and 130 ± 8 nM in TNF-α-treated RIN cells. The mean values of the magnitude of depolarization-induced increases in [Ca2+]i, i.e., Δ[Ca2+]i, were 1,212 nM (1,362 − 150 nM) in control cells and 2,093 nM (2,223 − 130 nM) in TNF-α-treated RIN cells. Therefore, there was a 73% greater increase in Δ[Ca2+]i in TNF-α-treated cells as compared to that in control RIN cells upon depolarization with 50 mM KCl.

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Figure 3. TNF-α enhanced the depolarization-induced increase in [Ca2+]i. The determination of [Ca2+]i by Fura-2 ratio fluorescence microscopy showed that upon addition of 50 mM KCl to control RIN cells, the [Ca2+]i increased from a basal value of 150 ± 9 to 1,362 ± 123 nM (n = 28), whereas in TNF-α-treated RIN cells, the corresponding values were 130 ± 8 (n = 20) and 2,223 ± 202 nM.

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With 5.5 μM ionomycin, TNF-α-treated RIN cells had a Δ[Ca2+]i of 2,795 ± 230 nM (n = 76 cells) compared to a Δ[Ca2+]i of 1,117 ± 165 nM (n = 58 cells) in control RIN cells (Fig. 4). The increase in [Ca2+]i was significantly greater (P = 0.010) in TNF-α-treated cells. TNF-α-treated RIN cells not only had higher [Ca2+]i levels, but the [Ca2+]i did not appear to have reached a plateau at this concentration of ionophore (5.5 μM) as it had in control cells. At lower concentrations of ionomycin (100 and 500 nM), the increase in [Ca2+]i tended to be lower in TNF-α-treated than in control RIN cells (Fig. 4). At 1.5 μM ionomycin, the increase in [Ca2+]i was similar in TNF-α-treated (524 ± 37 nM) and control (672 ± 64 nM) RIN cells.

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Figure 4. [Ca2+]i response to increasing concentrations of ionomycin. Concentrations of the ionophore are given as the final concentration in the medium. A: In control RIN cells, there was a marked increase in [Ca2+]i with 1.5 μM ionomycin. [Ca2+]i appeared to reach plateau at 5.5 μM ionomycin. B: In TNF-α-treated RIN cells, there was a marked increase in [Ca2+]i with 1.5 μM ionomycin similar to that seen in control cells. [Ca2+]i was greatly increased at 5.5 μM ionomycin and did not reach a plateau during the period of observation.

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Sixty-minute insulin release in response to carbachol was significantly increased in TNF-α-treated RIN cells (P < 0.011). Insulin release from TNF-α-treated RIN cells with carbachol was increased 206.4% ± 28.5% above TNF-α-treated RIN cells without carbachol (n = 8). Similarly, in control RIN cells, the increase in insulin secretion with carbachol was significant (P = 0.042) and its magnitude was 226.0% ± 43.8% (n = 7). There was no significant difference in insulin release with carbachol between TNF-α-treated RIN cells (20.63 ± 2.00 pg/mg protein/min) and control cells (19.35 ± 3.56 pg/mg protein/min).


Western blots of TNF-α-treated RIN cells revealed a decrease in calbindin-D28k levels (Fig. 5). Using ELISA for quantitation, there was a significant reduction in calbindin-D28k levels in TNF-α-treated RIN cells. By ELISA, TNF-α-treated cells had 175.2 ± 13.8 ng calbindin-D28k/mg protein (n = 4) compared with 331.5 ± 44.6 ng calbindin-D28k/mg protein (n = 4) in control cells. Therefore, treatment of RIN cells for 24 hr with TNF-α resulted in a significant (P = 0.016) reduction in calbindin-D28k levels.

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Figure 5. Immunoblot for calbindin-D28k in RIN cells. Western blot analysis of calbindin-D28k in TNF-α-treated (lane labeled T) and control cells (lane labeled C) revealed lower levels of calbindin-D28k (P = 0.016) in TNF-α-treated cells than in control cells. Lane 1 (leftmost lane) indicates marker proteins, whereas lane 4 (rightmost lane marked as S) is standard calbindin-D28k with molecular weight of 28.5 kDa.

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Proteins Regulating Apoptosis

We found levels of gene transcription for two members of the Bcl-2 family of apoptosis-regulating proteins to be unchanged as evaluated by RT-PCR. The intensity of bands for antiapoptotic Bcl-2 and proapoptotic Bax showed no obvious differences between TNF-α-treated and control RIN cells (Fig. 6). In a like manner, protein expression by Western blotting appeared to be similar for Bcl-2, Bax, as well as Bcl-xL, another inhibitor of apoptosis, in TNF-α-treated and control cells (Fig. 7).

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Figure 6. Effect of TNF-α on gene expression for Bcl-2 and Bax in RIN cells. Left: Lane 1 (leftmost lane marked as M), molecular weight markers; lane 2, Bcl-2 expression in control cells (C); lane 3, Bcl-2 expression in TNF-α-treated cells (T); lane 4, Bax expression in control RIN cells (C); lane 5, Bax expression in TNF-α-treated cells (T). No differences in levels of gene expression for Bcl-2 and Bax are seen. Right: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression in control (lane 2, labeled as C) and in TNF-α-treated (lane 3, labeled as T) cells and in kit control (lane 4, labeled as kC). cDNA was synthesized from 500 ng of total RNA using reverse transcriptase. One-tenth of the cDNA reaction was amplified for 40 cycles with Taq DNA polymerase with the specific primer sets.

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Figure 7. Effect of TNF-α on levels of protein expression for Bcl-2, Bax, and Bcl-xL in RIN cell. In each lane was loaded 30 μg of total protein as determined by Bio-Rad Dc microassay. Control RIN cells (lane 1) and TNF-α-treated RIN cells (lane 2) were similar in density for all three proteins.

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The confocal fluorescence microscopy data shown in Figure 8 indicate polarity in the distribution of Bcl-2 (Fig. 8A) and Bax (Fig. 8B) proteins in control RIN cells. Bcl-2 (shown in red; Fig. 8A) was distributed throughout the cell with more localization in the basal regions as shown by a histogram plot of mean integrated pixel intensities versus optical section number (Fig. 8C). The mean integrated pixel intensities described here were measured using Metamorph Version 4.6r5 software (Universal Imaging, PA) by drawing regions of interest in the cytoplasmic regions excluding the nuclei of the cells. The same regions of interest were also chosen for measuring the integrated pixel intensities for Bax distribution (shown in green; Fig. 8B) and the data shown in the histogram (Fig. 8D) indicate that in control RIN cells, Bax was localized more in the apical regions of cells. In TNF-α-treated RIN cells, Bcl-2 (Fig. 9A) was localized more in the basal and middle regions as opposed to the apical regions of the cells as measured by the mean integrated pixel intensities (Fig. 9C). In TNF-α-treated RIN cells, Bax localization (Fig. 9B) was greatest in the middle optical sections of the cells as measured by the mean integrated pixel intensities (Fig. 9D). The localization of the Bax and Bcl-2 proteins in TNF-α-treated RIN cells is associated with the distribution of intracellular organelles such as mitochondria and rough endoplasmic reticulum, which are typically concentrated in the perinuclear regions. The confocal microscopy data presented in Figure 10 indicate that TNF-α treatment resulted in colocalization of Bcl-2 and Bax predominantly in the perinuclear regions of the cells as shown by the distinct appearance of yellow color.

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Figure 8. Polarity in the distribution of Bcl-2 and Bax in control RIN cells. The confocal optical sections are displayed from the upper part of the cells (apical region; left), the middle part (middle), and the lower part of the cells, i.e., near the coverslip (basal region; right). A: Confocal optical sections of Bcl-2-immunoreacted control RIN cells. B: Confocal optical sections of Bax-immunoreacted control RIN cells. C: The histogram showing mean integrated pixel intensities of Bcl-2 immunoreacted with fluorescent-labeled secondary antibody in apical, middle, and basal regions of control RIN cells indicates Bcl-2 was localized more in the basal regions. D: The histogram showing mean integrated pixel intensities of Bax immunoreacted with fluorescent-labeled secondary antibody in apical, middle, and basal regions of control RIN cells indicates Bax was localized more in the apical regions. Scale bar = 5 μm.

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Figure 9. Polarity in the distribution of Bcl-2 and Bax in TNF-α-treated RIN cells. The confocal optical sections are displayed from the upper part of the cells (apical region; left), the middle part (middle), and the lower part of the cells, i.e., near the coverslip (basal region; right). A: Confocal optical sections of Bcl-2-immunoreacted TNF-α-treated RIN cells. B: Confocal optical sections of Bax-immunoreacted TNF-α-treated RIN cells. C: The histogram showing mean integrated pixel intensities of Bcl-2 immunoreacted with fluorescent-labeled secondary antibody in apical, middle, and basal regions of TNF-α-treated RIN cells. D: The histogram showing mean integrated pixel intensities of Bax immunoreacted with fluorescent-labeled secondary antibody in apical, middle, and basal regions of TNF-α-treated RIN cells. The intensity of immunoreactivity appears to be greatest in the perinuclear region for both proteins in the TNF-α-treated RIN cells. Scale bar = 3.5 μm.

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Figure 10. TNF-α-induced spatial interactions between Bcl-2 and Bax. A single confocal optical section of control (top) and TNF-α-treated (bottom) RIN cells dually labeled for Bcl-2 and Bax. TNF-α-treated cells (bottom) show a distinct yellow color in the merged image (right column), particularly in the perinuclear regions, thus indicating TNF-α-induced spatial interactions between the antiapoptotic protein Bcl-2 and the proapoptotic protein Bax in the RIN cells.

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Microarray Analysis

Gene expression profiling of TNF-α-treated RIN cells using two distinct microarray sets (Vanderbilt Microarray Shared Resources and Functional Genomics Core, University of Pennsylvania) revealed that six known genes were upregulated and nine known genes were downregulated (Table 1). Some of the proteins whose gene expression were upregulated by TNF-α included insulin-like growth factor 1A precursor (IGF-1A), protein tyrosine phosphatase receptor type A, sodium channel nonvoltage gated 1 gamma subunit Scnn 1g, ornithine decarboxylase antizyme inhibitor, Nbr1, and lysosome-associated membrane glycoprotein precursor LAMP-2. Similarly, the proteins whose gene expression was downregulated included vascular cell adhesion molecule-1 (VCAM-1), ras-related GTPase, a death-associated kinase 3, adenylate cyclase 3, and alcohol dehydrogenase. The approximately threefold downregulation of VCAM-1 (mean ratio was 0.36) and approximately fourfold (mean ratio was 4.1) upregulation of IGF-1A precursor were particularly interesting and are discussed below.

Table 1. Gene Expression Profiles of TNFα-treated RIN Cells
Murine mRNA Description Up regulatedMean Ratio*
  • *

    Data analysis of two experiments (one experiment with 4 array sets and the other with 2 sets were averaged).

Nbr1, complete cds4.2
Identical to 100% of lysosome-associated membrane glycoprotein 2 precursor LAMP-2 CD107B4.1
99% identity to 100% of insulin-like growth factor IA precursor, IGF-IA4.1
Protein tyrosine phosphatase, receptor type A2.0
Sodium channel nonvoltage-gated 1 gamma subunit Scnnlg1.9
Identical to 100% of ornithine decarboxylase antizyme inhibitor1.8
Down regulated 
Vascular cell adhesion molecule-1, complete cds0.36
Identical to 100% of ras-related GTPase rag, splice form A (human)0.497
Identical to 100% of NM_007828 death-associated kinase 3; ZIP kinase0.50
Villin 20.50
Identical to 100% of complement receptor CR1 precursor0.50
99% identity to 25% of XM_039712 adenylate cyclase 3 (human)0.52
Identical to 100% of AJ318416 N-WASP protein0.52
RIKEN cDNA 3110030G19 gene alcohol dehydrogenase PAN20.54
Identical to 100% of NM_026058 RIKEN cDNA 2900019C14 gene0.54


  1. Top of page
  2. Abstract
  6. Acknowledgements

In response to genetic and environmental stimuli, cells undergo programmed cell death known as apoptosis. Our goal was to determine the effect of TNF-α on β-cell apoptosis, replication, gene and protein expressions, including expression of calbindin-D28k and functions in insulin-producing and -secreting RIN cells. Neurotropic factor protection against neuronal cell death was highest with a 24-hr pretreatment at 10 ng/mL (Prehn et al., 1994); therefore, we selected 10 ng/mL as the concentration of TNF-α and 24 hr as the treatment period for this study. The results provide evidence for a signal transduction pathway in β-cells that connects TNF-α to decreased calbindin-D28k and subsequently to increased apoptosis, decreased proliferation, enhanced insulin secretion, and altered gene expression.

It is now well established that short-term elevations in [Ca2+]i in β-cells, e.g., Ca2+ transients and oscillations, are necessary and essential for β-cell function, including insulin secretion, but prolonged increases in [Ca2+]i can lead to deleterious conditions and even cell death (Pralong et al., 1990; Gilon et al., 1993). An increase in [Ca2+]i due to entry of extracellular Ca2+ and/or mobilization of Ca2+ from intracellular stores constitutes a major signal transduction pathway of apoptosis (Dowd, 1995; Thompson, 1995). Sustained increases in [Ca2+]i lead, inter alia, to activation of Ca2+-dependent proteases such as calpains, which belong to the superfamily of cysteine proteases that affect apoptosis (Thompson, 1995; Mattson et al., 1997a).

The signaling mechanisms for the proapoptotic and antiproliferative actions on the RIN cells in the present study are consistent with an effect of calbindin-D28k. Calbindin-D28k levels were decreased by 47% from control RIN cells upon TNF-α treatment (Fig. 5). TNF-α-treated RIN cells had a significantly greater (P = 0.01) increase in [Ca2+]i in response to addition of ionomycin at 5.5 μM than control cells. Similarly, depolarization with 50 mM KCl induced an increase in [Ca2+]i (i.e., Δ[Ca2+]i) that was 73% greater in TNF-α-treated RIN cells as compared to that in control RIN cells (Fig. 3). The decreased level of calbindin-D28k accompanied by increased [Ca2+]i would favor a proapoptotic state, since calbindin-D28k buffers intracellular Ca2+ (Rhoten and Sergeev, 1994; Reddy et al., 1997; Darios et al., 2003) and inhibits caspase-3 activity (Bellido et al., 2000; Christakos and Liu, 2004; Liu et al., 2004). The reduced amount of calbindin-D28k in the RIN cells would decrease the calbindin-D28k interacting specifically with caspase-3 (Palczewska et al., 2003), leading to an increase in caspase-3 activity and greater activity of downstream effectors. Taken together, the results indicate that the effect of TNF-α on increasing apoptosis is mediated at least in part by a reduction in calbindin-D28k and altered modulation of [Ca2+]i.

An abnormally elevated [Ca2+]i can be part of the apoptotic pathway, perhaps even triggering the apoptosis found in this study and in β-cells (Juntti-Berggren et al., 1993; Thompson, 1995). Rhoten and Sergeev (1994) showed that RIN cells with an increased content of calbindin-D28k maintained much more effectively than control cells a normal [Ca2+]i when challenged with potentially cell death-inducing increases in [Ca2+]i. Therefore, expression of calbindin-D28k in β-cells may enhance resistance to calcium-mediated apoptosis by direct and specific interactions with cell death mediators such as Ca2+ and caspase-3 (Palczewska et al., 2003) and help preserve β-cell function and delay the development of IDDM in the intact organism.

An important link between immune system-mediated diseases and levels of calbindin-D28k was established by Ho et al. (1996). The cytokines such as TNF-α produced by the islet-infiltrating macrophages and lymphocytes are involved in β-cell destruction (Mandrup-Poulsen et al., 1996; Rabinovitch and Suarez-Pinzon, 1998). Our results with RIN cells show more than a threefold increase in apoptotic β-cells. Increased apoptosis has been found in other β-cells treated with TNF-α (Ishizuka et al., 1999; Chang et al., 2003), indicating an apoptotic effect of TNF-α on β-cells.

Bcl-2 can protect cells from both calcium-induced and oxidative stress injury (Marin et al., 1996; Xie et al., 1996). The regulation of mitochondrial membrane integrity and the release of cytochrome c from mitochondria are important processes during apoptosis (Kroemer and Reed, 2000) and are controlled by the Bcl-2 family (Vander Heiden and Thompson, 1999), which also includes proapoptotic member Bax and antiapoptotic protein Bcl-xL. In response to apoptotic stimuli, the cytosolic proapoptotic members such as Bax plausibly redistribute to mitochondria, from the results presented in this study, and promote cytochrome c release either by forming a pore through oligomerization or by opening a channel called the voltage-dependent anion channel via direct interaction (Shimizu et al., 1999; Saito et al., 2000).

The activation of a dimeric transcription factor nuclear factor kappa B (NF-κB) by TNF-α can lead to expression of various antiapoptotic factors, which can then modulate the apoptotic actions of TNF-α (Lee et al., 1999; Wang et al., 1999). Recently, Papakonstanti and Stournaras (2004) showed that TNF-α by blocking caspase-3 activity acted as an antiapoptotic agent in opossum kidney cells. Such an action of TNF-α seems unlikely in the present study, where apoptosis was increased. Papakonstanti and Stournaras (2004) concluded that the inhibitory action of TNF-α on caspase-3 is mediated via actin redistribution through a novel signaling mechanism involving translocation of NF-κB to the nucleus. However, if the activation of the NF-κB pathway is somehow damaged or the protein synthesis is inhibited, the activation of TNF-α receptor 1 (TNF-R1) by TNF-α can cause apoptosis via the activation of caspase-8, which in turn activates caspase-3 (cf. Wei et al., 2001).

The impact of the heightened (fourfold increase) or suppressed (threefold decrease) transcripts such as IGF-1A precursor and VCAM-1 respectively on the observed effects of TNF-α remains to be determined. In a model of inflammatory bowel disease, Rutgeerts et al. (2003) showed that interaction between integrin α4β1 expressed on T-lymphocytes with the VCAM-1 on the endothelial cells resulted in transendothelial migration of lymphocytes (Baron et al., 1993). Interestingly, Piccarella et al. (1993) found that TNF-α transgenic mice had an elevated expression of VCAM-1 in islet endothelia, but the role of VCAM-1 in β-cells remains to be elucidated. The primary translation product of IGF-1A is an 18–19 kDa glycosylated propeptide (Lowe et al., 1988). The processing of this 18–19 kDa propeptide through a 12 kDa nonglycosylated intermediate may contribute IGF to the circulation in vivo. The IGFs with molecular weights of 7.5 kDa are a group of circulating peptides homologous to insulin in amino acid sequence and resemble preproinsulin in their tertiary structure and have insulin-like effects on tissues (Philips and Vassilopoulou-Sellin, 1980). The normal survival response to proinflammatory cytokines such as TNF-α is the increased expression of hormones such as IGF-1 (Kenchappa et al., 2004). IGFs are major contributors to the growth, maturation, and functions of β-cells and are expressed in islets throughout life (Hill and Hogg, 1992). IGF may protect cells by inhibiting induction of nitric oxide synthase (NOS) in some tissue types (Schini et al., 1994; Stewart and Rotwein, 1996; Hill et al., 1999). Several signaling mechanisms such as MAP kinase, c-Jun N-terminal kinase, and Akt have been implicated in the action of IGF. Further characterizing the regulatory pathways of IGF may provide molecular therapeutic targets for drug discovery for diabetes that will permit more specific therapies than the crude therapies being used at present. Therefore, much more research needs to be carried out on the specific signaling mechanisms affected by the changes in gene expression profiles found in this study.

In conclusion, it is exciting to consider the possibility of modulating the β-cell dysfunction of diabetes mellitus with new therapeutic strategies directed at knowledge gained on apoptosis, replication, transcription, calbindin-D28k, and regulation of intracellular Ca2+.


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  2. Abstract
  6. Acknowledgements

The authors acknowledge the Marshall University Genomics Core Facility for microarray analysis.


  1. Top of page
  2. Abstract
  6. Acknowledgements
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