Inhibition of mitochondrial gene transcription suppresses neurotensin secretion in the human carcinoid cell line BON
Nan Li,1
Qingding Wang,1
Jing Li,1
Xiaofu Wang,1
Mark R. Hellmich,1
Srinivasan Rajaraman,2
George H. Greeley, Jr.,1
Courtney M. Townsend, Jr.,1 and
B. Mark Evers1
Departments of 1Surgery and 2Pathology, The University of Texas Medical Branch, Galveston, Texas
Submitted 26 January 2004
; accepted in final form 3 September 2004
 |
ABSTRACT
|
---|
Mitochondria, organelles essential for ATP production, play a central role in a number of cellular functions, including the regulation of insulin secretion. Neurotensin (NT), an important regulatory intestinal hormone, has been implicated in fatty acid translocation, gut motility and secretion, and intestinal cell growth; however, mechanisms regulating NT secretion have not been entirely defined. The purpose of this study was to determine the effect of inhibition of mitochondrial gene transcription on NT secretion. BON cells, a novel human carcinoid cell line that produces and secretes NT peptide and expresses the gene encoding NT (designated NT/N), were treated with ethidium bromide (EB; 0.05, 0.1, and 0.4 µg/ml), an inhibitor of DNA and RNA synthesis, or vehicle over a time course (14 days). Cells were then stimulated with either ACh (100 µM) or phorbol 12 myristate,13-acetate (PMA, 10 nM) for 30 min. Media and cells were extracted, and NT peptide measured by RIA. Treatment with EB had no effect on BON cell viability or cell cycle distribution over the 4-day course. In contrast, EB treatment produced a dose-dependent reduction of mitochondrial gene expression; however, NT/N gene expression was not altered. Mitochondrial inhibition by EB treatment suppressed NT secretion induced by ACh and PMA, both in a dose-dependent manner. EB-mediated inhibition of NT secretion and mitochondrial gene expression was reversed with removal of EB. Our results demonstrate that inhibition of mitochondrial gene transcription suppresses both ACh- and PMA-stimulated NT release. These findings are the first to demonstrate that mitochondrial function is important for agonist-mediated NT secretion.
gut endocrine cells; peptide secretion
REGULATORY PEPTIDES OF THE gastrointestinal tract are localized to specialized endocrine cells of the gut mucosa that, in response to extracellular stimuli, act in an endocrine, paracrine, or autocrine fashion to coordinate and control numerous physiological functions related to digestion and nutrient absorption (9, 24). In addition to regulating motility, secretion, and blood flow, certain hormones regulate growth and gene expression in the gastrointestinal tract (reviewed in Refs. 39, 42, and 45). Analysis of the specific mechanisms that contribute to the regulation of gut peptide secretion is critical to our better understanding of the role of these peptides in gastrointestinal homeostasis. Despite the important role that gut peptides play in diverse physiological functions of the gastrointestinal tract, relatively little is known regarding the precise cellular mechanisms controlling gut hormone release, which is in direct contrast to hormones produced by pancreatic endocrine cells (e.g., insulin secretion in pancreatic
-cells) in which the mechanisms for hormone release have been well-characterized.
Mitochondria, the primary cellular organelles responsible for ATP production, are believed to be derived from genetic components of microorganisms and, therefore, exhibit a transcription and translation system that is, for the most part, separate from the nuclear genome (30). The mitochondrial genome of mammalian cells encodes 13 polypeptides, 2 ribosomal RNAs, and 22 transfer RNAs (51). Mitochondrial proteins, composed of enzyme complexes (I-IV), are involved in oxidative phosphorylation and cooperate with nuclear genome-encoded proteins for ATP production, which are required for vital cellular functions such as protein synthesis and vesicular transport (4, 48, 51). The role of mitochondria in pancreatic insulin release and exocytosis has been elucidated (2, 25, 3638, 40, 41, 44, 47). Mitochondrial dysfunctions resulting from alterations in the mitochondrial genome contribute to human diseases such as some cases of type II diabetes mellitus (6, 29, 35, 38, 49).
Ethidium bromide (EB) affects transcription/replication of extrachromosomal genes more specifically than those of chromosomal origin (11, 26, 54). Mitochondrial function has been examined in the insulin-secreting cell lines,
HC9, MIN6, and INS-1 (25, 30, 40, 44, 47). Low-dose EB effectively inhibited mitochondrial gene transcription with a concomitant inhibition of glucose-mediated insulin release; these effects were reversed upon removal of EB (25). Therefore, EB is a useful reagent to assess the role of mitochondrial function in peptide release. To date, no studies have specifically analyzed the role of mitochondrial function in gut hormone secretion.
Neurotensin (NT) is a gut peptide localized to specialized enteroendocrine cells (N cells) predominantly found in the distal small bowel (16, 23). NT stimulates pancreatic secretion and colonic motility, inhibits gastric and small bowel motility, facilitates fatty acid translocation from the intestinal lumen, and stimulates growth of various gastrointestinal tissues (1, 3, 5, 10, 19, 20, 28, 46, 52). Our laboratory is focused on better delineating the cellular mechanisms regulating the expression and secretion of NT. To determine the relationship between mitochondrial function and the stimulation of NT release, we utilized the BON cell line, which, similar to the terminally differentiated N cell, expresses high levels of the NT/neuromedin N (NT/N) gene and synthesizes and secretes NT peptide (8, 17, 22). In this report, we analyzed the effects of EB treatment on ACh- and phorbol ester [i.e., phorbol 12-myristate,13-acetate (PMA)]-stimulated NT secretion. Treatment with EB virtually eliminated mitochondrial transcription and significantly inhibited the effect of both ACh and PMA on stimulated NT release. These effects were reversed after removal of the EB. Importantly, our findings demonstrate that mitochondrial function contributes to stimulated gut peptide release.
 |
MATERIALS AND METHODS
|
---|
Materials.
EB was obtained from Amresco (Solon, OH). ACh, PMA, oligomycin, rotenone, BSA, pyruvate, uridine, propidium iodine, and the Cell Growth Determination Kit MTT Based were purchased from Sigma (St. Louis, MO). The NT enzyme immunoassay (EIA) kit was from Phoenix Pharmaceuticals (Belmont, CA). Cell culture reagents were from Fisher Scientific (Richardson, TX). [32P]dATP was from New England Nuclear Life Science Products (Boston, MA). QIAamp Blood Kit was from QIAGEN (Valencia, CA). ULTRASPEC RNA isolation system was from Biotecx Laboratories (Houston, TX). NorthernMax prehyb/hyb buffer was from Ambion (Austin, TX).
Cell culture.
The BON cell line was developed and characterized in our laboratory from a human pancreatic carcinoid tumor (18, 21). BON cells are maintained in a 1:1 mixture of DMEM and nutrient mixture, F-12K, supplemented with 5% FBS serum, 0.11 mg/ml pyruvate, and 0.05 mg/ml uridine, in 5% CO2 at 37°C. Cells reaching 7090% confluency were divided to a density of 5 x 104/cm2. For the treated cells, EB was added to the culture medium 1824 h after plating.
Cell growth and MTT assays.
For assessment of cell growth, BON cells were plated in 24-well culture plates at a density of 1 x 104/cm2 and counted by trypan blue exclusion using a hemocytometer. For MTT assays, BON cells were cultured in 96-well culture plates, and the assay was performed using the Cell Growth Determination Kit following the manufacturer's instructions. Spectrophotometric absorbance was measured at a wavelength of 570 nm using an Emax precision microplate reader (Molecular Devices, Sunnyvale, CA).
Flow cytometry.
BON cells were harvested with trypsin at various time points, washed two times with PBS, and resuspended in low-salt staining buffer containing 50 µg/ml propidium iodine, 30 mg/ml polyethylene glycol, 0.1% Triton X-100, and 4 mM sodium citrate. RNase A (100 µg/ml) was added, and the sample was incubated for 20 min at 37°C. An equal volume of high-salt-staining buffer containing 50 µg/ml propidium iodine, 30 mg/ml polyethylene glycol, 0.1% Triton X-100, and 400 mM sodium citrate was added, and the samples were stored at 4°C overnight. Cell cycle analysis was performed using a FACScan flow cytometer (Becton-Dickinson, San Jose, CA).
PCR for mitochondrial DNA.
BON cell total DNA was isolated and purified by a QIAamp Blood Kit. PCR was performed from 5 ng BON DNA using the following primers: 5'-GTGCAGCCGCTATTAAAGGT-3' and 5'-GCACCCCTCTGACATCC-3' (27). PCR conditions were as follows: 30 cycles for 1 min at 94°C, 1 min at 60°C, and 1 min at 72°C. Specific mitochondrial DNA probes were generated from the PCR products.
RNA isolation and Northern blot analysis.
Total cellular RNA was isolated by the ULTRASPEC RNA isolation system following the manufacturer's instructions. For Northern blot analysis, 20 µg total RNA were separated by formaldehyde-agarose (1.2%) gel electrophoresis and transferred to nitrocellulose membrane, as we have previously described (17). Blots were prehybridized in NorthernMax prehyb/hyb buffer for 4 h at 42°C. Hybridization was carried out overnight in the same buffer containing a 32P-labeled mitochondrial cDNA fragment (generated from above PCR products) or a human NT/N cRNA probe (7, 13). The filters were washed two times in 2x saline-sodium citrate (SSC), 0.1% SDS at room temperature, two times for 20 min in 0.1x SSC, 0.1% SDS at 42°C, and exposed to X-ray film. To correct for RNA loading, blots were stripped and reprobed with the constitutively expressed GAPDH gene. The intensities of signals on the autoradiogram were estimated by densitometric scanning, and the relative amount of each transcript was determined.
RIA for NT secretion and content.
The control and EB-treated cells were cultured in six-well plates. Each well was washed with secretion medium (DMEM/F-12K containing 1% dialyzed BSA) and preincubated for 30 min in secretion medium. ACh or PMA was then added in fresh media and incubated for 30 min. Medium was collected and stored at 20°C until RIA for NT. Cells were removed from culture plates by scraping and then sonicated in 1 M acetic acid containing 5 mM of EDTA, 1 mM phenylmethlylsulfonyl fluoride, and 100 U/ml aprotinin. After being boiled for 5 min, cell extracts were centrifuged at 20,000 g for 20 min at 4°C. Supernatants were saved for NT assays. The NT content of the medium and cell extracts was determined by RIA, as described previously (33, 53). NT secretion was determined as follows:
Relative NT secretion was given as the degree of increase corrected by controls. Data presented are means ± SE (n = 3).
For the treatment of BON cells with the mitochondrial inhibitors oligomycin and rotenone, NT content in the medium was determined by the EIA kit following the manufacturer's instructions.
Statistical analysis.
Data in Fig. 1 were analyzed using the Kruskal-Wallis test because of heterogeneous variability in each group. Data from Fig. 5 and the reversible changes (see Fig. 7) were analyzed by ANOVA for a one-way classification. Fisher's least-significant difference procedure was used for multiple comparisons with Bonferroni adjustment for the number of comparisons. Data from the NT secretion time study (see Fig. 6) were analyzed using the two-sample t-test to compare treatment with 0.1 µg/ml EB and treatment without EB for each day separately. All tests were assessed at the 0.05 level of significance.

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 1. Inhibition of oligomycin on phorbol 12-myristate,13-acetate (PMA)-mediated neurotensin (NT) secretion from BON cells. BON cells were serum-starved in secretion medium overnight. Cells were pretreated with 0.8 µg/ml oligomycin or 10 µM rotenone for 30 min and then treated with PMA with the combination of oligomycin or rotenone for another 30 min. The medium was collected, and NT content in the medium was measured by an enzyme immunoassay (EIA) kit following the manufacturer's instruction. Experiments were performed in triplicate. Real NT concentrations were compared among groups. Results are expressed as means ± SE (n = 4 experiments). P < 0.05 vs. control (). *P < 0.05 vs. PMA.
|
|

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 5. Effect of EB on NT secretion. BON cells were treated with various concentrations of EB for 2 days. Either 100 µM ACh (A) or 10 nM PMA (B) was added for 30 min. RIA was used to measure NT peptide content in BON cells and in the media. EB treatment inhibited NT secretion induced by ACh and PMA in a dose-dependent manner. *P < 0.05 vs. ACh (100 µM). P < 0.05 vs. PMA (10 nM).
|
|

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 7. Reversible change of mitochondrial gene expression after EB treatment in BON cells. A: BON cells were treated with various concentrations of EB for 3 days. The medium was replaced by EB-free medium, and the total RNA was prepared at the indicated times. Northern blot analysis probed for mitochondrial gene expression; the blot was stripped and reprobed with GAPDH. The left lane shows the total RNA from control cells cultured for 3 days. B: densitometric analysis of the Northern blot shown in A. The amounts of mitochondrial RNA were normalized by GAPDH expression. After removal of EB from the medium, mitochondrial gene expression was gradually restored from 1 to 5 days at EB dosages of 0.05 and 0.1 µg/ml; however, cells treated with 0.4 µg/ml EB showed no recovery. C: BON cells were treated with 0.1 µg/ml EB for 3 days. The medium was replaced with EB-free medium and cultured for the indicated times. NT secretion was induced with 100 µM ACh and was measured by RIA, as described in MATERIALS AND METHODS. Secretion of NT, in response to ACh, was partially restored at 4 and 6 days after removal of EB. *P < 0.05 vs. ACh (100 µM, EB-free day 2).
|
|

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 6. Time course analysis of NT secretion in EB-treated cells. BON cells were treated with 0.1 µg/ml EB for 14 days. Either 100 µM ACh (A) or 10 nM PMA (B) was added for 30 min. NT content of the cell extract and media was measured by RIA. The inhibitory effects of EB treatment on NT secretion, after stimulation with ACh or PMA, were gradually increased from 1 to 4 days. *P < 0.05 vs. ACh (100 µM). P < 0.05 vs. PMA (10 nM).
|
|
 |
RESULTS
|
---|
Oligomycin, a mitochondrial inhibitor, inhibits PMA-mediated NT secretion from BON cells.
Oligomycin, a potent inhibitor of mitochondrial ATP synthase (14), inhibits insulin secretion in pancreatic
-cells stimulated by K+ or tolbutamide (43). To first assess the potential role of mitochondria in NT secretion from BON cells, we pretreated BON cells with oligomycin (0.8 µg/ml) in secretion medium for 30 min and with PMA (10 nM) combined with oligomycin for another 30 min. The medium was collected for NT secretion measured by the NT EIA kit. As we have previously reported, NT secretion is increased in BON cells treated with PMA (33, 34); pretreatment with oligomycin significantly decreased NT release (Fig. 1). These findings suggest that acute inhibition of mitochondrial function can decrease NT secretion from BON cells. Another mitochondrial inhibitor, rotenone, which is an inhibitor of mitochondrial electron transport (14), was also used; NT secretion from BON cells was decreased but did not achieve statistical significance (data not shown). Together, these findings suggest that the acute inhibition of mitochondrial function may inhibit stimulated NT peptide release from the BON cell line.
Effect of EB on cell proliferation, cell viability, and cell cycle distribution.
To further establish the potential importance of mitochondria for gut peptide release, we established a chronic model of mitochondrial gene suppression in BON cells using EB treatment, as previously done in pancreatic
-cell models (25). First, to examine the temporal profiles of mitochondrial gene expression and NT secretion after EB treatment, we first determined the effects of EB on BON cell proliferation, cell viability, and cell cycle distribution. BON cells were treated with various concentrations of EB and harvested at the specified time points. BON cell proliferation was not altered significantly by EB treatment, even up to 4 days (Fig. 2A). However, 6 days after EB treatment, the growth of BON cells was significantly inhibited with the indicated concentrations of EB. There was also no effect on cell viability by EB treatment up to 4 days (Fig. 2B). Flow cytometric analysis showed that 60% of untreated BON cells were in G0/G1 phase and 21% of cells were in S phase of the cell cycle on day 4. Moreover, there was no significant change in cell cycle distribution in BON cells treated with EB (Fig. 2C). These results demonstrate no effect on the proliferation, viability, and cell cycle distribution of BON cells by treatment with EB at a concentration range of 0.050.4 µg/ml over the 4-day time course. Therefore, the remainder of our studies analyzing the effect of mitochondrial inhibition on NT secretion and NT/N gene expression was performed over the 4-day treatment period.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 2. Effect of ethidium bromide (EB) on cell proliferation, cell viability, and cell cycle. BON cells were treated with various concentrations of EB and harvested at indicated time points. A: cell number was counted using a hemocytometer. B: cell viability was measured by MTT (shown on day 4). C: cell cycle distribution was determined by DNA flow cytometric analysis (shown on day 4). Cell proliferation, cell viability, and cell cycle distribution were not significantly altered after treatment with various concentrations of EB for up to 4 days.
|
|
Inhibition of mitochondrial gene expression after EB treatment.
To determine the effect of EB on mitochondrial gene expression in BON cells, we incubated cells for 3 days with various concentrations of EB. Total RNA was isolated, and mitochondrial expression was determined by Northern blot analysis (Fig. 3A). Densitometric analysis shows mitochondrial gene expression levels decreased to 27, 8, and 3% of control cells with EB concentrations of 0.05, 0.1, and 0.4 µg/ml, respectively, normalized by expression levels of GAPDH on the same blot (Fig. 3B). The mitochondrial gene transcript that we measured in our study was the 16S mitochondrial ribosomal RNA (1.6-kb size) using a probe design previously described by Heddi et al. (27).

View larger version (42K):
[in this window]
[in a new window]
|
Fig. 3. Effect of EB on mitochondrial gene expression. BON cells were incubated for 3 days with various concentrations of EB, and RNA was isolated. A: changes in mitochondrial (Mt) gene expression in EB-treated cells were measured by Northern blot analysis. For our studies, we analyzed the 16S mitochondrial rRNA (1.6 kb) using a probe described by Heddi et al. (27). B: densitometric analysis of the Northern blot shown in A. The amount of mitochondrial RNA was normalized to levels of GAPDH expression. Mitochondrial gene expression decreased in a dose-dependent manner after EB treatment. Results represent findings from a single representative experiment.
|
|
To further investigate the time course effects of EB on the expression levels of mitochondrial and nuclear genes, we treated BON cells with 0.4 µg/ml EB, and total RNA was isolated at the indicated times. The changes of the expression levels of mitochondrial and NT/N genes were measured by Northern blot analysis. Equal loading of RNA was normalized by expression levels of GAPDH (Fig. 4). The inhibitory effects increased gradually from day 1 to day 4 after EB treatment (Fig. 4A). Densitometric analysis shows that mitochondrial gene expression levels of the control cells were not significantly altered, whereas those of EB-treated cells were inhibited from 35 to 97% compared with control cells from day 1 to day 4 (Fig. 4B). In contrast, expression of the NT/N gene, which transcribes to two mRNA species (1.0- and 1.5-kb sizes) as a result of distinct polyadenylation sites (12, 15, 17, 32), was not significantly affected with EB treatment (Fig. 4, A and B). Expression of the "housekeeping" gene, GAPDH, was also unaffected by EB treatment (Fig. 4, A and B). These results demonstrate that treatment of EB at up to 0.4 µg/ml for 4 days reduced mitochondrial transcription without affecting the expression of nuclear genes, suggesting that, similar to the findings of others (11, 26, 54), EB inhibits replication/expression of mitochondria genes more significantly than nuclear genes.

View larger version (45K):
[in this window]
[in a new window]
|
Fig. 4. Time course analysis of expression of nuclear and mitochondrial genes in EB-treated BON cells. BON cells were treated with 0.4 µg/ml EB, and total RNA was isolated at the indicated time. A: changes of gene expression during EB treatment were measured by Northern blot analysis. B: densitometric analysis of the Northern blot shown in A. The amount of mitochondrial RNA was normalized to levels of GAPDH expression. These analyses demonstrated that, on days 14, mitochondrial RNA expression levels of the control cells were not significantly altered, whereas those of EB-treated cells were inhibited from 35 to 97% compared with control cells. In contrast, expression of the NT/N gene demonstrated no inhibition during EB treatment.
|
|
Suppression of agonist-mediated NT secretion along with inhibition of mitochondrial gene expression by EB treatment.
BON cells represent an ideal model for determining mechanisms of NT secretion. Previous studies have shown that ACh and PMA induce NT secretion in BON cells (33, 53). Because EB treatment at 0.4 µg/ml for 4 days inhibited mitochondrial gene replication and expression, and produced mitochondrial morphological changes without affecting nuclear gene expression and replication, we treated BON cells with EB at a concentration range of 0.050.4 µg/ml for 2 days to determine whether mitochondrial function is involved in agonist-mediated NT secretion. After 100 µM ACh or 10 nM PMA treatments for 30 min, NT peptide content in BON cells or in the media was measured by RIA. NT secretion was increased significantly in BON cells after treatments with 100 µM ACh or 10 nM PMA for 30 min (Fig. 5). ACh-induced NT secretion was inhibited from
2- to 4-fold (Fig. 5A), and PMA-induced NT secretion was inhibited from
8- to 13-fold after EB treatment (Fig. 5B). EB treatment inhibited ACh- or PMA-induced NT secretion in a dose-dependent manner.
As shown in Fig. 3, inhibition of expression levels of mitochondrial genes occurred from the first day after EB treatment, and the inhibitory effects of EB increased gradually from day 1 to day 4. To further investigate the relationship between mitochondria dysfunction and the inhibition of NT secretion, we performed a time course analysis of NT secretion in EB-treated cells (Fig. 6). BON cells were treated with 0.1 µg/ml EB for 14 days after 100 µM ACh or 10 nM PMA treatments for 30 min. The inhibitory effects of EB treatment on ACh-mediated NT secretion were observed on days 24 after EB treatment (Fig. 6A). In contrast, PMA-mediated NT secretion was inhibited only on day 2 after EB treatment (Fig. 6B). However, of note, BON cells not treated with EB were less responsive to PMA treatment after 2 days in culture, which may reflect alterations associated with cell confluency. In contrast, ACh-mediated NT secretion, in BON cells not treated with EB, was unchanged over the 4-day culture period.
Reversible change of mitochondrial gene expression after EB treatment.
To determine whether inhibition of mitochondrial gene expression could be reversed, we treated BON cells with various concentrations of EB for 3 days. The medium was replaced by EB-free medium, and total RNA was prepared at the indicated times. Northern blot analysis was performed for mitochondrial gene expression; mitochondrial RNA expression was normalized by GAPDH expression (Fig. 7). After removal of EB from the medium, mitochondrial gene expression was gradually restored over the 5-day course from 2379% and 661% at EB dosages of 0.05 and 0.1 µg/ml, respectively (Fig. 7, A and B). However, cells treated with 0.4 µg/ml EB showed no recovery (Fig. 7, A and B). These results demonstrate that the inhibitory effects of EB treatment on mitochondrial gene expression in BON cells was partially reversed using concentrations of 0.050.10 µg/ml.
To further determine the role of mitochondria on agonist-mediated NT secretion, we next determined whether inhibition of NT secretion by EB treatment could be recovered along with restoration of mitochondrial gene expression. BON cells were treated with 0.1 µg/ml EB for 3 days. The medium was replaced with EB-free medium and cultured for the indicated times. NT secretion was induced with 100 µM ACh and measured by RIA. Secretion of NT, in response to ACh, was partially restored at 4 and 6 days after removal of EB (Fig. 7C).
 |
DISCUSSION
|
---|
In our present study, we have blocked mitochondrial gene transcription by EB treatment to delineate the role of mitochondrial function on stimulated NT secretion in the BON endocrine cell line. This cell line, established from a human pancreatic carcinoid tumor (18, 21), demonstrates properties identical to the specialized N cell of the small intestine (8). In this regard, BON cells have provided an invaluable model to analyze mechanisms regulating endocrine gene expression and peptide release (17, 22, 31, 33, 50, 53). We demonstrate that chronic EB treatment at a dosage of 0.100.40 µg/ml blocked mitochondrial gene expression without altering nuclear gene transcription. Moreover, in EB-treated BON cells, NT release was decreased significantly after treatment with either ACh or PMA, which have both been shown to potently stimulate NT release (33, 53). Removal of EB reversed the alterations in mitochondrial gene transcription and, similarly, the effect of ACh on NT secretion was restored.
EB, which is known to intercalate into double-strand nucleic acids, is a useful reagent for the analysis of extrachromosomal genetic components because it specifically inhibits mitochondrial gene transcription (11, 26, 54). After culture in sublethal EB concentrations, cells lack mitochondrial DNA and its transcripts, thus effectively abolishing mitochondrial function and allowing an accurate analysis of the contribution of mitochondria to selected cellular processes, such as peptide release and exocytosis. The sensitivity to EB appears to be cell-type-specific, with cell death occurring over a fairly wide concentration range. In BON cells treated with EB, mitochondrial gene expression decreased in a dose-dependent fashion, with undetectable levels noted at a dose of 0.100.40 µg/ml. Furthermore, at this dosage of EB, BON cells remained viable, and cell cycle parameters were not altered over a 4-day treatment period. Therefore, for further analysis of NT secretion, we assessed BON cells treated with EB at this dose range in which mRNAs encoded by mitochondrial DNA were completely depleted and no changes in cell growth or viability were observed.
Insulin secretion from pancreatic
-cells has been examined using various inhibitors and stimulators of mitochondrial function (2, 25, 3638, 40, 41, 44, 47). With the use of the pancreatic
-cell line MIN6, depleted of mitochondrial DNA by EB treatment, Tsuruzoe et al. (47) confirmed impaired ATP production through the mitochondrial oxidative phosphorylation pathway and, moreover, showed that insulin secretion, in response to either glucose or leucine, was inhibited in the EB-treated cells. In contrast, arginine-induced insulin secretion was not impaired, indicating that mitochondrial ATP production was required for certain secretagogues (i.e., glucose and leucine) but not for others (e.g., arginine). In addition, MIN6 cells depleted of mitochondrial DNA showed a relatively weak response to the sulfonylurea drug glibenclamide, suggesting that ATP production is also important for the stimulation of insulin secretion induced by the sulfonylureas, which are commonly used to treat patients with type II diabetes mellitus.
In our current study, we show that EB treatment, at a dosage that inhibits BON cell mitochondrial gene expression, significantly altered the release of NT in response to ACh and PMA. Similar to pancreatic
-cells and stimulated insulin release, these findings suggest that mitochondrial function is important for agonist-mediated NT peptide release from the BON endocrine cell line. At least with the secretagogues used in this study, the response was similar with either ACh or PMA treatment. Removal of EB effectively reversed the suppression of mitochondrial gene transcription and the inhibition of ACh-induced NT release. These findings indicate that the changes observed with EB treatment were a result of mitochondrial dysfunction and that the treatment produced no irreversible effects on the chromosomal genes and the secretory capacities of BON cells. In fact, recovery of NT secretion correlated with the restoration of mitochondrial gene expression.
In conclusion, we demonstrate that EB treatment of the BON endocrine cell line effectively suppresses mitochondrial gene transcription without significantly altering cell growth or nuclear gene transcription, thus providing a unique model to analyze the contribution of mitochondrial function on NT peptide release. In addition, these effects were shown to be reversible with cessation of EB treatment. Our results show that mitochondria are important, but not solely responsible, for secretagogue-induced NT release. Importantly, our study is the first to suggest a role for mitochondria in agonist-induced gut peptide release. Finally, this novel model system can be used to address additional questions regarding the mechanism of regulatory peptide release in gut endocrine cells.
 |
GRANTS
|
---|
This work was supported by National Institutes of Health Grants R37 AG-10885, PO1 DK-35608, and RO1 DK-48345.
 |
ACKNOWLEDGMENTS
|
---|
We thank Tatsuo Uchida for statistical analysis and Eileen Figueroa and Karen Martin for manuscript preparation.
 |
FOOTNOTES
|
---|
Address for reprint requests and other correspondence: B. Mark Evers, Dept. of Surgery, The Univ. of Texas Medical Branch, 301 Univ. Boulevard, Galveston, TX 77555-0536 (E-mail: mevers{at}utmb.edu)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
 |
REFERENCES
|
---|
- Andersson S, Rosell S, Hjelmquist U, Chang D, and Folkers K. Inhibition of gastric and intestinal motor activity in dogs by (Gln4) neurotensin. Acta Physiol Scand 100: 231235, 1977.[ISI][Medline]
- Antinozzi PA, Ishihara H, Newgard CB, and Wollheim CB. Mitochondrial metabolism sets the maximal limit of fuel-stimulated insulin secretion in a model pancreatic beta cell: a survey of four fuel secretagogues. J Biol Chem 277: 1174611755, 2002.[Abstract/Free Full Text]
- Armstrong MJ, Parker MC, Ferris CF, and Leeman SE. Neurotensin stimulates [3H]oleic acid translocation across rat small intestine. Am J Physiol Gastrointest Liver Physiol 251: G823G829, 1986.[Abstract/Free Full Text]
- Attardi G and Schatz G. Biogenesis of mitochondria. Annu Rev Cell Biol 4: 289333, 1988.[ISI][Medline]
- Baca I, Feurle GE, Schwab A, Mittmann U, Knauf W, and Lehnert T. Effect of neurotensin on exocrine pancreatic secretion in dogs. Digestion 23: 174183, 1982.[ISI][Medline]
- Ballinger SW, Shoffner JM, Hedaya EV, Trounce I, Polak MA, Koontz DA, and Wallace DC. Maternally transmitted diabetes and deafness associated with a 10.4 kb mitochondrial DNA deletion. Nat Genet 1: 1115, 1992.[ISI][Medline]
- Bean AJ, Dagerlind A, Hokfelt T, and Dobner PR. Cloning of human neurotensin/neuromedin N genomic sequences and expression in the ventral mesencephalon of schizophrenics and age/sex matched controls. Neuroscience 50: 259268, 1992.[CrossRef][ISI][Medline]
- Carraway RE, Mitra SP, Evers BM, and Townsend CM Jr. BON cells display the intestinal pattern of neurotensin/neuromedin N precursor processing. Regul Pept 53: 1729, 1994.[CrossRef][ISI][Medline]
- Chung DH and Evers BM. The digestive system. In: The Physiologic Basis of Surgery, edited by O'Leary JP. Baltimore MD: Lippincott, Williams & Wilkins, 2002, p. 457490.
- Chung DH, Evers BM, Shimoda I, Townsend CM Jr, Rajaraman S, and Thompson JC. Effect of neurotensin on gut mucosal growth in rats with jejunal and ileal Thiry-Vella fistulas. Gastroenterology 103: 12541259, 1992.[ISI][Medline]
- Desjardins P, Frost E, and Morais R. Ethidium bromide-induced loss of mitochondrial DNA from primary chicken embryo fibroblasts. Mol Cell Biol 5: 11631169, 1985.[ISI][Medline]
- Dobner PR, Barber DL, Villa-Komaroff L, and McKiernan C. Cloning and sequence analysis of cDNA for the canine neurotensin/neuromedin N precursor. Proc Natl Acad Sci USA 84: 35163520, 1987.[Abstract]
- Dong Z, Wang X, Zhao Q, Townsend CM Jr, and Evers BM. DNA methylation contributes to expression of the human neurotensin/neuromedin N gene. Am J Physiol Gastrointest Liver Physiol 274: G535G543, 1998.[Abstract/Free Full Text]
- Duchen MR and Biscoe TJ. Relative mitochondrial membrane potential and [Ca2+]i in type I cells isolated from the rabbit carotid body. J Physiol 450: 3361, 1992.[Abstract]
- Evers BM. Expression of the neurotensin/neuromedin N gene in the gut. In: Gastrointestinal Endocrinology, edited by Greeley GH Jr. Totowa, NJ: Humana, 1999, p. 425438.
- Evers BM. Endocrine gene neurotensin: molecular mechanisms and a model of intestinal differentiation. World J Surg 26: 799805, 2002.[CrossRef][ISI][Medline]
- Evers BM, Ishizuka J, Townsend CM Jr, Rajaraman S, and Thompson JC. Expression of neurotensin messenger RNA in a human carcinoid tumor. Ann Surg 214: 448455, 1991.[ISI][Medline]
- Evers BM, Ishizuka J, Townsend CM Jr, and Thompson JC. The human carcinoid cell line, BON. A model system for the study of carcinoid tumors. Ann NY Acad Sci 733: 393406, 1994.[ISI][Medline]
- Evers BM, Izukura M, Chung DH, Parekh D, Yoshinaga K, Greeley GH, Jr., Uchida T, Townsend CM Jr, and Thompson JC. Neurotensin stimulates growth of colonic mucosa in young and aged rats. Gastroenterology 103: 8691, 1992.[ISI][Medline]
- Evers BM, Izukura M, Townsend CM Jr, Uchida T, and Thompson JC. Neurotensin prevents intestinal mucosal hypoplasia in rats fed an elemental diet. Dig Dis Sci 37: 426431, 1992.[ISI][Medline]
- Evers BM, Townsend CM Jr, Upp JR, Allen E, Hurlbut SC, Kim SW, Rajaraman S, Singh P, Reubi JC, and Thompson JC. Establishment and characterization of a human carcinoid in nude mice and effect of various agents on tumor growth. Gastroenterology 101: 303311, 1991.[ISI][Medline]
- Evers BM, Wang X, Zhou Z, Townsend CM Jr, McNeil GP, and Dobner PR. Characterization of promoter elements required for cell-specific expression of the neurotensin/neuromedin N gene in a human endocrine cell line. Mol Cell Biol 15: 38703881, 1995.[Abstract]
- Ferris CF, Carraway RE, Hammer RA, and Leeman SE. Release and degradation of neurotensin during perfusion of rat small intestine with lipid. Regul Pept 12: 101111, 1985.[CrossRef][ISI][Medline]
- Guyton AC. Secretory functions of the alimentary tract. In: Textbook of Medical Physiology, edited by Guyton AC. Philadelphia, PA: Saunders, 1991, p. 709724.
- Hayakawa T, Noda M, Yasuda K, Yorifuji H, Taniguchi S, Miwa I, Sakura H, Terauchi Y, Hayashi J, Sharp GW, Kanazawa Y, Akanuma Y, Yazaki Y, and Kadowaki T. Ethidium bromide-induced inhibition of mitochondrial gene transcription suppresses glucose-stimulated insulin release in the mouse pancreatic beta-cell line betaHC9. J Biol Chem 273: 2030020307, 1998.[Abstract/Free Full Text]
- Hayashi J, Tanaka M, Sato W, Ozawa T, Yonekawa H, Kagawa Y, and Ohta S. Effects of ethidium bromide treatment of mouse cells on expression and assembly of nuclear-coded subunits of complexes involved in the oxidative phosphorylation. Biochem Biophys Res Commun 167: 216221, 1990.[ISI][Medline]
- Heddi A, Lestienne P, Wallace DC, and Stepien G. Mitochondrial DNA expression in mitochondrial myopathies and coordinated expression of nuclear genes involved in ATP production. J Biol Chem 268: 1215612163, 1993.[Abstract/Free Full Text]
- Izukura M, Evers BM, Parekh D, Yoshinaga K, Uchida T, Townsend CM Jr, and Thompson JC. Neurotensin augments intestinal regeneration after small bowel resection in rats. Ann Surg 215: 520527, 1992.[ISI][Medline]
- Kadowaki T, Kadowaki H, Mori Y, Tobe K, Sakuta R, Suzuki Y, Tanabe Y, Sakura H, Awata T, Goto Y, Hayakawa T, Matsuoka K, Kawamori R, Kamada T, Horai S, Nonaka I, Hagura R, Akanuma Y, and Yazaki Y. A subtype of diabetes mellitus associated with a mutation of mitochondrial DNA. N Engl J Med 330: 962968, 1994.[Abstract/Free Full Text]
- Kennedy ED, Maechler P, and Wollheim CB. Effects of depletion of mitochondrial DNA in metabolism secretion coupling in INS-1 cells. Diabetes 47: 374380, 1998.[Abstract]
- Kim M, Cooke HJ, Javed NH, Carey HV, Christofi F, and Raybould HE. D-glucose releases 5-hydroxytryptamine from human BON cells as a model of enterochromaffin cells. Gastroenterology 121: 14001406, 2001.[ISI][Medline]
- Kislauskis E, Bullock B, McNeil S, and Dobner PR. The rat gene encoding neurotensin and neuromedin N. Structure, tissue-specific expression, and evolution of exon sequences. J Biol Chem 263: 49634968, 1988.[Abstract/Free Full Text]
- Li J, Hellmich MR, Greeley GH Jr, Townsend CM Jr, and Evers BM. Phorbol ester-mediated neurotensin secretion is dependent on the PKC-
and -
isoforms. Am J Physiol Gastrointest Liver Physiol 283: G1197G1206, 2002.[Abstract/Free Full Text]
- Li J, O'Connor KL, Hellmich MR, Greeley GH Jr, Townsend CM Jr, and Evers BM. The role of protein kinase D in neurotensin secretion mediated by protein kinase C-
/-
and Rho/Rho kinase. J Biol Chem 279: 2846628474, 2004.[Abstract/Free Full Text]
- Maassen JA and Kadowaki T. Maternally inherited diabetes and deafness: a new diabetes subtype. Diabetologia 39: 375382, 1996.[CrossRef][ISI][Medline]
- Maechler P. Novel regulation of insulin secretion: the role of mitochondria. Curr Opin Investig Drugs 4: 11661172, 2003.[Medline]
- Maechler P and Wollheim CB. Mitochondrial signals in glucose-stimulated insulin secretion in the beta cell. J Physiol 529: 4956, 2000.[Abstract/Free Full Text]
- Maechler P and Wollheim CB. Mitochondrial function in normal and diabetic beta-cells. Nature 414: 807812, 2001.[CrossRef][ISI][Medline]
- Merchant JL, Dickinson CJ, and Yamada T. Molecular biology of the gut: model of gastrointestinal hormones. In: Physiology of the Gastrointestinal Tract, edited by Johnson LR, Alpers DH, and Cristensen J. New York: Raven, 1994, p. 295350.
- Noda M, Yamashita S, Takahashi N, Eto K, Shen LM, Izumi K, Daniel S, Tsubamoto Y, Nemoto T, Iino M, Kasai H, Sharp GW, and Kadowaki T. Switch to anaerobic glucose metabolism with NADH accumulation in the beta-cell model of mitochondrial diabetes. Characteristics of betaHC9 cells deficient in mitochondrial DNA transcription. J Biol Chem 277: 4181741826, 2002.[Abstract/Free Full Text]
- Patterson GH, Knobel SM, Arkhammar P, Thastrup O, and Piston DW. Separation of the glucose-stimulated cytoplasmic and mitochondrial NAD(P)H responses in pancreatic islet beta cells. Proc Natl Acad Sci USA 97: 52035207, 2000.[Abstract/Free Full Text]
- Reubi JC. Peptide receptors as molecular targets for cancer diagnosis and therapy. Endocr Rev 24: 389427, 2003.[Abstract/Free Full Text]
- Rustenbeck I, Herrmann C, and Grimmsmann T. Energetic requirement of insulin secretion distal to calcium influx. Diabetes 46: 13051311, 1997.[Abstract]
- Soejima A, Inoue K, Takai D, Kaneko M, Ishihara H, Oka Y, and Hayashi JI. Mitochondrial DNA is required for regulation of glucose-stimulated insulin secretion in a mouse pancreatic beta cell line, MIN6. J Biol Chem 271: 2619426199, 1996.[Abstract/Free Full Text]
- Thomas RP, Hellmich MR, Townsend CM Jr, and Evers BM. Role of gastrointestinal hormones in the proliferation of normal and neoplastic tissues. Endocr Rev 24: 571599, 2003.[Abstract/Free Full Text]
- Thor K and Rosell S. Neurotensin increases colonic motility. Gastroenterology 90: 2731, 1986.[ISI][Medline]
- Tsuruzoe K, Araki E, Furukawa N, Shirotani T, Matsumoto K, Kaneko K, Motoshima H, Yoshizato K, Shirakami A, Kishikawa H, Miyazaki J, and Shichiri M. Creation and characterization of a mitochondrial DNA-depleted pancreatic beta-cell line: impaired insulin secretion induced by glucose, leucine, and sulfonylureas. Diabetes 47: 621631, 1998.[Abstract]
- Tzagoloff A and Myers AM. Genetics of mitochondrial biogenesis. Annu Rev Biochem 55: 249285, 1986.[CrossRef][ISI][Medline]
- Van den Ouweland JM, Lemkes HH, Ruitenbeek W, Sandkuijl LA, de Vijlder MF, Struyvenberg PA, van de Kamp JJ, and Maassen JA. Mutation in mitochondrial tRNA(Leu)(UUR) gene in a large pedigree with maternally transmitted type II diabetes mellitus and deafness. Nat Genet 1: 368371, 1992.[ISI][Medline]
- Von Wichert G, Jehle PM, Hoeflich A, Koschnick S, Dralle H, Wolf E, Wiedenmann B, Boehm BO, Adler G, and Seufferlein T. Insulin-like growth factor-I is an autocrine regulator of chromogranin A secretion and growth in human neuroendocrine tumor cells. Cancer Res 60: 45734581, 2000.[Abstract/Free Full Text]
- Wallace DC. Mitochondrial genetics: a paradigm for aging and degenerative diseases? Science 256: 628632, 1992.[ISI][Medline]
- Wood JG, Hoang HD, Bussjaeger LJ, and Solomon TE. Neurotensin stimulates growth of small intestine in rats. Am J Physiol Gastrointest Liver Physiol 255: G813G817, 1988.[Abstract/Free Full Text]
- Zhang T, Townsend CM Jr, Udupi V, Yanaihara N, Rajaraman S, Beauchamp RD, Ishizuka J, Evers BM, Gomez G, Thompson JC, and Greeley GH Jr. Phorbol ester-induced alteration in the pattern of secretion and storage of chromogranin A and neurotensin in a human pancreatic carcinoid cell line. Endocrinology 136: 22522261, 1995.[Abstract]
- Zylber E, Vesco C, and Penman S. Selective inhibition of the synthesis of mitochondria-associated RNA by ethidium bromide. J Mol Biol 44: 195204, 1969.[ISI][Medline]
Copyright © 2005 by the American Physiological Society.