help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2004-0285
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow A correction has been published
Right arrow All Versions of this Article:
145/9/4408    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, L.
Right arrow Articles by Jiang, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, L.
Right arrow Articles by Jiang, C.
Endocrinology Vol. 145, No. 9 4408-4414
Copyright © 2004 by The Endocrine Society

Disruption of Glucose Sensing and Insulin Secretion by Ribozyme Kir6.2-Gene Targeting in Insulin-Secreting Cells

Li Li, Asheebo Rojas, Jianping Wu and Chun Jiang

Department of Biology, Georgia State University, Atlanta, Georgia 30302-4010

Address all correspondence and requests for reprints to: Dr. Chun Jiang, Department of Biology, Georgia State University, 24 Peachtree Center Avenue, Atlanta, Georgia 30302-4010. E-mail: cjiang{at}gsu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The ATP-sensitive K+ (KATP) channel, composed of Kir6.2 and sulfonylurea receptor (SUR1), in pancreatic ß-cells is believed to serve as a metabolic sensor regulating insulin secretion according to glucose levels. Thus, genetic disruption of Kir6.2 expression may impair KATP channel function in glucose sensing and insulin secretion. Here we show evidence obtained from functional genetic assays supporting this hypothesis. To avoid adaptive cellular mechanisms in transgenic preparations, we designed a hammerhead ribozyme that specifically targeted the Kir6.2 mRNA at serine 78. The Kir6.2-ribozyme was constructed in an adenoviral vector and expressed in insulin-secreting RINm5F cells. Both RT-PCR and Northern blot analyses showed that Kir6.2 transcripts were significantly reduced with a Kir6.2-ribozyme treatment. Whole-cell patch-clamp studies indicated that the Kir6.2-ribozyme treatment lowered KATP channel density by 66%. In response to higher glucose challenge, insulin release from the RINm5F cells dropped by approximately 20% in a transfection dose of 0.7 multiplicity of infection, and by 30–40% in a dose of 2.7 multiplicity of infection. These results therefore indicate that KATP channels play an important role in glucose sensing and insulin secretion, and ribozyme Kir6.2-gene targeting is an effective approach for selective inhibition of functional expression of KATP channels.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ATP-SENSITIVE K+ (KATP) CHANNELS regulate cellular excitability according to intracellular ATP levels, a property that may allow them to serve as a glucose sensor in pancreatic ß-cells (1, 2, 3). The KATP channels are composed of the pore-forming Kir6.2 subunit and the regulatory sulfonylurea receptor (SUR1) in the ß-cells (4, 5). Several previous studies have demonstrated that mutations of either of these two subunits affect insulin secretion from pancreatic ß-cells and predispose a number of diseases including diabetes and persistent hyperinsulinemic hypoglycemia of infancy (6, 7, 8, 9, 10, 11, 12). Transgenic mice expressing ß-cell KATP channels with reduced ATP sensitivity develop severe hyperglycemia, hypoinsulinemia, and ketoacidosis within 2 d after birth and typically die within 5 d (2, 10). Knockout of the Kir6.2 gene in mice totally eliminates the glucose-induced depolarization, Ca2+ influx, and insulin secretion (2, 8).

Although the transgenic and gene-knockout preparations have greatly advanced the understanding of KATP channel functions, several drawbacks have seriously limited applications of these techniques. For instance, such techniques are time-consuming and labor-intensive (13). Sometimes it is impossible to distinguish what produces the phenotype, as the observed systemic consequence can be a result of a loss of function of the targeted gene or a compensatory response of the organism to the lost gene (14). Also, genomic manipulations of certain genes can cause embryonic lethality that prevents establishment of transgenic animals and assessment of gene functions (15). In contrast, these problems can be largely avoided with the catalytic ribozyme techniques that provide an effective alternative in current functional genomic studies (16, 17).

To assess the function of KATP channels in glucose sensing and insulin secretion, we performed experiments by disrupting the pore-forming Kir6.2 subunit using the hammerhead ribozyme-based gene targeting. The effects of the ribozyme treatment on Kir6.2 mRNA, functional KATP channels, and insulin secretion in insulin-secreting RINm5F cells were studied.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction and cloning of Kir6.2-ribozyme in an adenovirus expression vector
A hammerhead ribozyme, called Kir6.2-ribozyme, targeting the GUC triplets at position 233 of the Kir6.2 nucleotide sequence with respect to the first AUG (GenBank accession no. D50581) was constructed according to previously described procedures (18, 19). The sequence of the Kir6.2-ribozyme contained a 22-bp domain of the hammerhead ribozyme (20) and a 12-bp sequence complementary to the Kir6.2 mRNA (Fig. 1Go, A and B). The cleavage site was placed at Ser78 in the Kir6.2 peptide chain. An EcoRI restriction site was added to each end of Kir6.2-ribozyme for an insertion into the gateway vector ENTR1A (Invitrogen Life Technologies, Carlsbad, CA).



View larger version (59K):
[in this window]
[in a new window]
  		FIG. 1. A, The Kir6.2-ribozyme was designed to cleave Kir6.2 mRNA at position 233 referring to the start codon or Ser78 in the amino acid sequence as indicated. Note that there are 131 nucleotides before the first ATG in the 5'UTR. B, The Kir6.2 mRNA (lowercase letters) was aligned with the antisense ribozyme sequence (uppercase letters). C, Kir6.2-ribozyme and GFP control plasmid were expressed in the transfected RINm5F cells at a dose of 8 x 106 pfu/dish. The presence of Kir6.2-ribozyme was confirmed by RT-PCR analysis in which a 142-bp fragment was observed in the cells transfected with the Kir6.2-ribozyme (lane 2), but not in those transfected with the GFP control plasmid (lane 3). D–G, Greenfluorescence was observed in RINm5F cells 1–7 d post transfection. The highest level of expression was seen on d 3 post transfection. On d 3, most cells showed positive GFP expression when observed under fluorescent (H) and transparent (I) light. Calibration in D and H, 100 µm.

 
A template 5'-TCGGAATTCCGAGGAAGCTGATGAGTCCGTGAGGACGAAACA TGGCGGAATTCCGTA nucleotide and a primer 5'-TACGGAATTCCGCCATG were synthesized by Sigma-Genosys (www.sigma-genosys.com), with which the PCR was performed to produce a double-strand DNA fragment coding for the Kir6.2-ribozyme. The PCR products were subjected to electrophoresis on a 1.7% agarose gel, and the 57-bp product was excised. The secondary structure was then analyzed using the RNA Mfold program developed by Dr. Michael Zuker (www.bioinfo.rpi.edu/~zukerm) to confirm that the nucleotide sequence at position 233 was accessible for ribozyme cleavage.

By homologous recombination, the Kir6.2-ribozyme was transferred from the gateway vector ENTR1A to the adenovirus expression plasmid pAd/CMV/V5-DEST (Invitrogen Life Technologies). The presence of the Kir6.2-ribozyme in the pAd/CMV/V5-DEST vector was confirmed by DNA sequencing and restriction mapping. Similar methods were used to construct the control vector containing green fluorescence protein (GFP) inserted into the ENTR1A vector via two restriction sites, i.e. KpnI and NotI.

Cell culture
The HEK-293A cell line was purchased from Invitrogen Life Technologies, and the RINm5F cells were obtained from American Type Culture Collection (Manassas, VA). The cells were cultured in the DMEM and RPMI 1640 medium, respectively, in the presence of 10% fetal calf serum, 1% penicillin/streptomycin, and 2% glucose. The cells were grown in a humidified incubator containing 5% CO2 at 37 C and were split using 0.05% trypsin-0.02% EDTA (vol/vol) when they became 90% confluent.

Preparation of viral lysate
The amplification of adenoviral stock was produced according to the manufacturer’s instruction (Invitrogen Life Technologies). The pAd/CMV/V5-DEST plasmid with Kir6.2-ribozyme was digested with the PacI endonuclease to expose the inverted terminal regions. HEK-293A cells at 90% confluence were transfected with the digested plasmid using Lipofectamine 2000 (Invitrogen Life Technologies). The cytopathic effect was observed 10–12 d post transfection when the cells were surrendered from further studies. The ribozyme-containing cells were harvested by squirting cells off the dish. To prepare crude viral lysate, cells and media were transferred to a sterile 15-ml capped tube and subjected to several freeze-thaw cycles, followed by centrifugation at 3000 rpm for 15 min. The viral stock was then placed at –80 C. Control studies were performed using the pAd/CMV/V5-DEST vector alone or this vector with the GFP sequence.

Transfection of RINm5F cells with the adenoviral stock
The titer of adenoviral stock was determined by estimating plaque-forming units (pfu) after the transfection of HEK293 cells (21). The titers of the Kir6.2-ribozyme plasmid, GFP control plasmid, and vector alone were about 1 x 108 pfu/ml. RINm5F cells (~3 x 106 cells/dish) were treated with two different doses of the Kir6.2-ribozyme plasmid [2 x 106and 8 x 106 pfu/dish, corresponding to multiplicities of infection (moi) of 0.7 and 2.7, respectively]. Similarly, RINm5F cells were transfected with the GFP control plasmid or vector only in 8 x 106 pfu/dish. The transfected cells were incubated at 37 C for 24 h to allow the expression of Kir6.2-ribozyme and GFP.

Northern blot analysis
The total RNA was isolated from RINm5F cells treated with the Kir6.2-ribozyme and pAd/CMV/V5-DEST vector alone with the RNeasy kit as recommended by the supplier (Qiagen, Valencia, CA; see online Fig. 1Go). About 3 x 106 cells were used for each preparation. The riboprobe (202 bp) for the Kir6.2 mRNA was designed to cover a fragment of the 5'-untranslated region (5'UTR) beginning 119 bp upstream of the start codon (ATG) and ending 83 bp in the open reading frame of the Kir6.2 transcript, in which the lowest sequence similarity to other inward rectifier K+ channels was found. The single-strand riboprobe was synthesized using an in vitro transcription kit (Roche, Indianapolis, IN) and was labeled with alkaline phosphatase-coupled digoxigenin (Roche).

For Northern blot analysis, 20 µg total RNA (denatured at 65 C for 10 min) were electrophoresed on a 1.5% agarose gel containing 5% formaldehyde. Equal amounts of RNA were used in different samples, which was further confirmed by equal ethidium bromide stains. The total RNA was blotted onto a nylon membrane (Roche) and then exposed to 50 ng/µl riboprobe. Hybridization was performed in 250 mM sodium phosphate buffer containing 50–60% formamide, 250 mM NaCl, and 7% (vol/vol) sodium dodecyl sulfate (SDS) at 68 C for 16 h (pH 7.4). Transcript size calculation was based on the RNA marker (Invitrogen Life Technologies). After hybridization was complete, the membrane was washed twice (10 min each time) at room temperature in low stringency wash buffer (2x standard saline citrate and 0.1% SDS), and then twice (20 min each time) at 68 C in high stringency wash buffer (0.5x standard saline citrate and 0.1% SDS). The membrane was visualized with substrates nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate of the alkaline phosphatase.

RT-PCR analysis
Total RNA (4 µg) treated with 2 U deoxyribonuclease at 37 C for 15 min was used to synthesize first-strand cDNAs using random hexamer primers in a 20-µl reaction system. The presence of Kir6.2-ribozyme in the transfected cells was detected with PCR. One primer (5'-GTCCTCACGGACTCATC) targeted at the consensus sequence of the ribozyme, and the other bound to the sequence of the T7 promoter in the pAd/CMV/V5-DEST vector. The PCR products were determined by electrophoresis to be approximately 142 bp.

For detection of the cleavage efficiency of the Kir6.2-ribozyme, another PCR was performed to amplify a 483-bp fragment with a forward primer (5'-ATGCTGTCCCGAAAAGG CTTATCCCTGAGG) and a reverse primer (5'-GGCGTTGATCATTAGCCCTACGATATTC TGC). The PCR products scaling amino acids from the first methionine to Ala161 were analyzed on a 1.0% agarose gel and visualized with ethidium bromide. To assess the variation in first-strand cDNAs, a 842-bp fragment of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified as an internal control.

Whole-cell patch-clamp
Whole-cell voltage-clamp recording was performed at room temperature as described previously (22, 23). In brief, fire-polished patch pipettes were made from 1.2 mm borosilicate capillary glass (Sutter Instruments, Novato, CA). Tight seals (>1 G{Omega} before breaking into the whole-cell mode) were obtained with the transfected cells. The patch electrodes had an open tip resistance of 0.5–1.0 M{Omega}. The series resistance during recording varied from 5–10 M{Omega} and was not compensated. Recordings were terminated whenever a significant increase (>20%) in series resistance occurred. Current records were low pass filtered (2 kHz; Bessel; four-pole filter, –3 dB), digitized (20 kHz, 16-bit resolution), and stored on computer disk for later analysis using the pCLAMP 8 software (Axon Instruments, Foster City, CA). Recordings were performed using solutions containing equal concentrations of K+ applied to the bath and recording pipettes. This solution contained 125 mM potassium gluconate, 10 mM KCl, 1 mM MgCl2, 5 mM EGTA, 2 mM ATP (K+ salt), 10 mM D-glucose, and 10 mM HEPES (pH 7.4). ATP was omitted in the bath solution. Levcromakalim (10 µM) and glibenclamide (10 µM) dissolved in dimethylsulfoxide were sequentially applied to the bath solution.

Insulin secretion
RINm5F cells treated or untreated with Kir6.2-ribozyme were cultured in RPMI 1640 medium. After removal of culture medium, cells were washed twice using a glucose-free Krebs-Ringer bicarbonate (KRB) buffer, followed by a 30-min preincubation with KRB buffer at 37 C. To avoid the loss of cells during sequential supplements with different glucose concentrations, each dish was stimulated with only one glucose concentration for insulin measurement. Insulin secretion was assayed after incubation for 60 min at 37 C using KRB buffer supplemented with 2.8, 5.5, 10.0, 16.7, or 25.0 mM glucose. After glucose stimulation was finished, the medium was collected and centrifuged for 10 min at 5000 rpm, and the supernatant was frozen at –20 C for insulin measurement. The cells were digested with 0.05% trypsin-0.02% EDTA (vol/vol) and centrifuged at 5000 rpm for 5 min. The total cellular DNA was extracted from each dish using the DNeasy tissue kit (Qiagen). Quantitative insulin measurement was conducted using a rat insulin ELISA (Mercodia, Uppsala, Sweden). The insulin concentration was normalized by the DNA levels and expressed as nanograms per microgram of DNA per 60 min.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of Kir6.2-ribozyme in insulin-secreting RINm5F cells
RINm5F cells were transfected with Kir6.2-ribozyme using an adenoviral stock. RINm5F cell morphology did not show any evident change over a period of 1 wk post transfection. The presence of the Kir6.2-ribozyme in RINm5F cells was confirmed with RT-PCR showing a band at the expected size of 142 bp that was not found in control groups transfected with a GFP-containing plasmid (Fig. 1CGo) or the expression vector alone (not shown). When the ribozyme was replaced with the GFP sequence, strong green fluorescence was observed under the fluorescent microscope (Fig. 1Go, D–G). Stable expression of GFP was seen between d 2 and 5. On d 3, more than 90% of cells showed GFP fluorescence without abnormalities in cell morphology (Fig. 1Go, H and I), indicating that the adenovirus expression vector is highly effective and does not produce apparent cytotoxicity.

Inhibition of Kir6.2 expression in RINm5F cells
To determine whether the Kir6.2-ribozyme really produced a disruption of Kir6.2 transcripts, we performed Northern blot and RT-PCR analyses. Experiments were performed on RINm5F cells 3 d post transfection with the Kir6.2-ribozyme. Northern blot analysis of ribozyme function is based upon the cleavage site, i.e. if the ribozyme works effectively, the Kir6.2 mRNA should be cleaved into two fragments at position 233 referring to the start codon, producing two bands of 364 and 1122 bp, respectively (Fig. 2AGo). Otherwise, only one band of the full-length (1486 bp) would be seen. Our riboprobe contains the 5'UTR covering from –119 to 83 bp, so that only sequence within this range can bind to the riboprobe and be detected. In fact, the 364-bp fragment and the 1486-bp undigested fragment were stained in RINm5F cells treated with the Kir6.2-ribozyme in 8 x 106 pfu/dish, both of which contained the riboprobe binding sequence. The 1122-bp fragment was not shown because of the lack of riboprobe binding sequence. The 364-bp band was much stronger than the 1486 bp band, although the molecular mass of the cleaved mRNA was much smaller, suggesting that most Kir6.2 mRNA is digested (Fig. 2BGo). In contrast, only one 1486-bp band was present in vector-treated control cells (Fig. 2BGo).



View larger version (45K):
[in this window]
[in a new window]
  		FIG. 2. Analysis of Kir6.2 mRNA expression in RINm5F cells transfected with Kir6.2-ribozyme. A, Schematic of the cleaved Kir6.2 mRNA with indications of each fragment. The riboprobe used in the Northern analysis covered both the coding sequence and 5'UTR. Primers 1 and 2 used in RT-PCR analysis were expected to lead to a 483-bp PCR product when Kir6.2 mRNA was intact. B, Northern blot analysis revealed two bands of about 360 and 1,500 bp in the ribozyme-treated cells (lane 1) and only one band of approximately 1500 bp in cells treated with vector alone (lane 2). Note that the 360-bp band has a higher intensity than the 1500-bp band in the left lane. C, RT-PCR analysis showed a 483-bp band in cells treated with vector alone (lane 6). Such a band became less intense in cells exposed to the Kir6.2-ribozyme in a dose of 2 x 106 pfu/dish (lane 7) and 8 x 106 pfu/dish (lane 8). Control experiments were performed with GAPDH showing a 842-bp fragment that remained the same under above conditions (lanes 2–4). Control experiments were also performed by looking at DNAs without reverse transcriptase. The lack of stain indicates that all DNAs are degraded by deoxyribonuclease, and there is no DNA contamination in the RT-PCR system (lanes 5 and 9).

 
Consistent with the Northern blot analysis, RT-PCR showed a clear difference in Kir6.2 expression levels between the treated and untreated RINm5F cells. The intensity of the 483-bp fragment was lower in the presence of Kir6.2-ribozyme 1–4 d post transfection than in the control cells (Fig. 2CGo), whereas the 842-bp fragment of internal standard GAPDH remained unchanged in two groups of RINm5F cells with and without Kir6.2-ribozyme treatment (Fig. 2CGo).

Reduction of KATP channel density in Kir6.2-ribozyme-treated RINm5F cells
If the Kir6.2 mRNA is disrupted by the Kir6.2-ribozyme, Kir6.2 gene expression should decrease, leading to a reduction in the density of functional KATP channels in cellular membranes. To test this idea, we studied channel density using whole-cell patch-clamp in the RINm5F cells. Because KATP channels are normally inhibited at physiological concentrations of ATP, we used 10 µM levcromakalim, a KATP channel activator, to reveal the KATP currents. Levcromakalim exposure increased the inward rectifying currents by 536.0 ± 81.8% (n = 12) in RINm5F cells treated with the pAd/CMV/V5-DEST vector alone (Fig. 3AGo). At the maximal current activation, 10 µM glibenclamide, a KATP channel blocker, suppressed the inward rectifying currents to the baseline level (99.4 ± 13.8%; n = 21; Fig. 3AGo). Kir6.2-ribozyme treatment greatly reduced the currents affected by levcromakalim and glibenclamide (Fig. 3BGo). The difference in the maximum vs. minimum currents produced by the KATP channel activator and blocker was normalized by the whole-cell capacitance and used as an index of KATP channel density. Kir6.2-ribozyme treatment markedly reduced channel density, although no significant changes in the baseline currents were found before levcromakalim and glibenclamide exposures. Compared with that of untreated cells, the channel density of Kir6.2-ribozyme treated cells was lowered by approximately 66% [258 ± 27 (n = 12) vs. 88 ± 9 pA/pF (n = 9); Fig. 3CGo].



View larger version (18K):
[in this window]
[in a new window]
  		FIG. 3. A, Whole-cell currents were recorded from a RINm5F cell using a bath solution containing 140 mM K+. K+ currents with weak inward rectification were seen in the cell 3 d after a transfection with 8 x 106 pfu/dish of the vector alone. The currents were strongly activated by 10 µM levcromakalim and were inhibited by 10 µM glibenclamide. B, In another cell treated with the Kir6.2-ribozyme, currents affected by this KATP channel activator and blocker were much smaller, although the baseline currents were similar. C, KATP channel density was calculated as: ID = (ILev – IGlib)/WC, where ID is the channel density, ILev is the levcromakalim-sensitive current, IGlib is the glibenclamide-sensitive current, and WC is the whole-cell membrane capacitance. The channel density in ribozyme-transfected cells was 66% less than that in control cells.

 
Suppression of insulin secretion from RINm5F cells by Kir6.2-ribozyme
If KATP channels play a role in insulin secretion, the cleavage of Kir6.2 transcripts by the Kir6.2-ribozyme should affect insulin release from the insulin-secreting RINm5F cells. Thus, quantitative analysis of insulin secretion from RINm5F cells was carried out after glucose stimulation using rat insulin ELISA (24). The cells were treated with the vector alone or the Kir6.2-ribozyme at 0.7 and 2.7 moi, respectively. The amount of insulin was expressed as nanograms per microgram of DNA per 60 min. Insulin secretion at low concentrations of glucose (2.8 and 5.5 mM) was barely affected by 0.7 or 2.7 moi Kir6.2-ribozyme treatment (Table 1Go). With glucose concentrations of 10.0, 16.7, and 25.0 mM, however, treatment with 0.7 moi Kir6.2-ribozyme produced evident suppression of the insulin level (Fig. 4AGo). Stronger suppression was observed after treatment with a higher dose of Kir6.2-ribozyme (2.7 moi; Fig. 4AGo). In addition to the dose dependence, the reduction of insulin release was a function of exposure time (Fig. 4BGo). Maximal suppression of insulin secretion occurred 2–3 d post transfection and started declining after 4 d.


View this table:
[in this window]
[in a new window]
  		TABLE 1. ELISA analysis of insulin secretion from RINm5F cells treated with vector alone and Kir6.2-ribozyme

 


View larger version (19K):
[in this window]
[in a new window]
  		FIG. 4. Glucose-induced insulin secretion from RINm5F cells. Quantitative analysis of insulin released by the RINm5F cells was performed using insulin ELISA. The insulin level in each cell culture was normalized with the total DNA amount in each dish (nanograms per microgram of DNA per 60 min). A, The percent inhibition of insulin secretion was calculated for ribozyme-treated RINm5F cells after the cells were exposed to different glucose levels for approximately 60 min at 37 C. Slight or significant suppression of insulin secretion occurred with a ribozyme treatment of 2 x 106 pfu/dish (0.7 moi; P < 0.01). Stronger inhibition of insulin release was found with a higher dose of ribozyme treatment (8 x 106 pfu/dish or 2.7 moi) after glucose challenges of 10.0, 16.7, and 25.0 mM. Insulin secretion showed a slight increase with 2.8 mM glucose after ribozyme treatment. Data were obtained from average of consecutive 4-d measurements and are presented as the mean ± SE. B, The time profile shows that ribozyme treatment (2.7 moi) produced evident inhibition of insulin secretion on d 1, reached the maximal level on d 2–3, and started declining on d 4 of glucose stimulation at 10.0, 16.7, and 25.0 mM. No evident changes in insulin secretion were seen with 2.8 and 5.5 mM.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have developed a hammerhead ribozyme that specifically cleaves serine 78 of Kir6.2 mRNA. The ribozyme was used to disrupt Kir6.2 transcripts in insulin-secreting RINm5F cells. Our systematic analysis indicates that the Kir6.2-ribozyme is effective in terms of the disruption of Kir6.2 mRNA, the reduction in functional KATP channels, and the suppression of insulin release from RINm5F cells.

Understanding of the function of KATP channels in the regulation of cellular excitability and insulin secretion was greatly advanced after molecular cloning of the Kir6.2 and SUR1 subunits (4, 5), especially after Kir6.2- and SUR1-gene targetings were carried out in mice (2, 7, 8, 10, 11). Kir6.2-transgenic and Kir6.2-null mice show abnormalities in glucose sensing, insulin secretion, and ß-cell morphology (2, 7, 8, 10, 11), supporting the idea that KATP channels play an important role in ß-cells (25, 26, 27). Interestingly, Kir6.2 genetically engineered mice do not spontaneously develop diabetes even though the glucose-sensing mechanism in ß-cells is seriously impaired (2, 8, 10), suggesting that systemic compensation mechanisms have taken place in these Kir6.2 genetically engineered mice.

The catalytic ribozyme provides an alternative approach to the gene targeting of ß-cell KATP channels. Unlike the genomic approaches, the ribozyme-based gene targeting affects the cells in a short period, which may not allow systemic compensatory mechanisms to take over the lost gene (17). Also, it is much easier to construct the ribozyme vector than to produce transgenic mice, making this approach broadly available for functional genomics. The ribozyme is capable of binding the targeted gene specifically in a sequence-dependent manner and cleaving the target mRNAs only, leading to the inhibition of specific gene expression (17, 28). With the establishment of its efficiency and safety, therapeutic applications for the ribozymes have been attempted in the treatment of cancer and infectious diseases (29, 30).

The hammerhead ribozyme is the smallest ribozyme allowing a wide range of manipulations with available molecular genetic techniques (31, 32). The Kir6.2-ribozyme was designed to specifically and repetitively cleave the Kir6.2 mRNAs at Ser78 and thus breaks the Kir6.2 transcript into two pieces. Using the Mfold program (see Materials and Methods), we have identified several potential cutting sites for the hammerhead ribozyme. Among them, the targeting site at serine 78 showed the least possible secondary structures and thus was chosen in the present study. The disrupted Kir6.2 mRNAs should not produce functional channels, as each of the segments has only one transmembrane domain, assuming that they still could be translated (33, 34, 35). Indeed, our systematic analysis of Kir6.2-ribozyme effects on Kir6.2 mRNAs, functional KATP channels, and insulin secretion from the RINm5F cells indicates that the ribozyme-mediated mRNA disruption is effective and directly proportional to the dose given to the cells. In addition to these technical contributions, our functional assays also show evidence for the role of the KATP channels in insulin-secreting cells and provide additional support to the current theory that the KATP channels act as glucose sensors in pancreatic ß-cells (2, 3).

Because our Kir6.2 gene targeting was designed by taking advantage of the adenoviral expression system that has been widely applied in gene delivery in vivo (36, 37, 38), it is possible to apply the Kir6.2-ribozyme to cultured pancreatic ß-cells and whole-body animals. It is known that the RINm5F cells that were developed from insulinoma are not real ß-cells and may have different glucose-sensing and insulin secretion properties. Before the Kir6.2-ribozyme can be practiced in native ß-cells, therefore, two technical issues need to be addressed. First, the maximal inhibition of the targeted genes is far from complete. We observed about 60–70% inhibition of functional KATP channels in single cells treated with the ribozyme. The inhibition of insulin secretion was even lower, ranging from 30–40%. Second, the stable expression of the Kir6.2-ribozyme only lasted 3–4 d in RINm5F cells. Such a narrow window may not allow extensive functional assessments of a gene in whole-body animals. However, these problems should not be overemphasized. Incomplete suppression of the targeted genes is a common characteristic of ribozyme gene targeting as previously documented (39). Although functional assays may require higher levels of suppression of gene expressions, a complete knockout of certain genes may seriously affect cellular activity, which can hinder the assessment of functions of the targeted genes. In this regard, ribozyme-based gene targeting remains an effective approach to knock down, rather than knock out, these genes. Also, there may be another way to overcome the narrow window for stable Kir6.2-ribozyme expression, such as repetitively administering the ribozyme to cells, finding other expression vectors, etc. Clearly, further studies are needed to achieve stable and long-term expression of higher levels of ribozymes in both in vitro and in vivo systems.

In conclusion, we have developed a hammerhead ribozyme targeted at the Kir6.2 mRNAs. Our systematic analysis of its effects on disruption of the Kir6.2 mRNAs, inhibition of KATP channel expression in plasma membranes, and suppression of glucose-induced insulin release in RINm5F cells indicates that this is a promising tool in Kir6.2 gene targeting. These studies also support the idea that KATP channels function as glucose sensors in pancreatic ß-cells, and the proper number of pancreatic ß-cells should be retained to regulate blood glucose levels. We believe that additional studies along this line of research can lead to the design of better approaches to gene targeting for functional genomic assays and more effective therapeutic modalities for the treatment of diabetes and other diseases that remain challenging to modern medicine.


   Acknowledgments
 
Special thanks to Na Wei and Jean-Pierre Muhumuza for their technical assistance. We are grateful to SmithKline Beecham Pharmaceuticals for the gift of levcromakalim (BRL38227).


   Footnotes
 
This work was supported by National Institutes of Health Grants HL-58410 and HL-67890 and the American Diabetes Association (1-01-RA-12).

Abbreviations: GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescence protein; KATP, ATP-sensitive K+; KRB, Krebs-Ringer bicarbonate; moi, multiplicity of infection; pfu, plaque-forming unit; SDS, sodium dodecyl sulfate; SUR1, sulfonylurea receptor; UTR, untranslated region.

Received March 5, 2004.

Accepted for publication May 20, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Ashcroft FM, Gribble FM 1998 Correlating structure and function in ATP-sensitive K+ channels. Trends Neurosci 21:288–294[CrossRef][Medline]
  2. Seino S, Iwanaga T, Nagashima K, Miki T 2000 Diverse roles of KATP channels learned from Kir6.2 genetically engineered mice. Diabetes 49:311–318[Abstract]
  3. Nichols CG, Koster JC 2002 Diabetes and insulin secretion: whither KATP? Am J Physiol 283:E403–E412
  4. Aguilarbryan L, Nichols CG, Wechsler SW, Clement JP, Boyd AE, Gonzalez G, Herrerasosa H, Nguy K, Bryan J, Nelson DA 1995 Cloning of the ß-cell high-affinity sulfonylurea receptor: a regulator of insulin-secretion. Science 268:423–426[Abstract/Free Full Text]
  5. Inagaki N, Gonoi T, Clement JP, Namba N, Inazawa J, Gonzalez G, Aguilar-Bryan L, Seino S, Bryan J 1995 Reconstitution of IKATP: an inward rectifier subunit plus the sulfonylurea receptor. Science 270:1166–1170[Abstract/Free Full Text]
  6. Thomas PM, Cote GJ, Wohllk N, Haddad B, Mathew PM, Rabl W, Aguilarbryan L, Gagel RF, Bryan J 1995 Mutations in the sulfonylurea receptor gene in familial persistent hyperinsulinemic hypoglycemia of infancy. Science 268:426–429[Abstract/Free Full Text]
  7. Miki T, Tashiro F, Iwanaga T, Nagashima K, Yoshitomi H, Aihara H, Nitta Y, Gonoi T, Inagaki N, Miyazaki J, Seino S 1997 Abnormalities of pancreatic islets by targeted expression of a dominant-negative KATP channel. Proc Natl Acad Sci USA 94:11969–11973[Abstract/Free Full Text]
  8. Miki T, Nagashima K, Tashiro F, Kotake K, Yoshitomi H, Tamamoto A, Gonoi T, Iwanaga T, Miyazaki J, Seino S 1998 Defective insulin secretion and enhanced insulin action in KATP channel-deficient mice. Proc Natl Acad Sci USA 95:10402–10406[Abstract/Free Full Text]
  9. Otonkoski T, Ammala C, Huopio H, Cote GJ, Chapman J, Cosgrove K, Ashfield R, Huang E, Komulainen J, Ashcroft FM, Dunne MJ, Kere J, Thomas PM 1999 A point mutation inactivating the sulfonylurea receptor causes the severe form of persistent hyperinsulinemic hypoglycemia of infancy in Finland. Diabetes 48:408–415[Abstract]
  10. Koster JC, Marshall BA, Ensor N, Corbett JA, Nichols CG 2000 Targeted overactivity of ß cell KATP channels induces profound neonatal diabetes. Cell 100:645–654[CrossRef][Medline]
  11. Seghers V, Nakazaki M, DeMayo F, Aguilar-Bryan L, Bryan J 2000 Sur1 knockout mice: a model for KATP channel-independent regulation of insulin secretion. J Biol Chem 275:9270–9277[Abstract/Free Full Text]
  12. Gloyn AL, Weedon MN, Owen KR, Turner MJ, Knight BA, Hitman G, Walker M, Levy JC, Sampson M, Halford S, McCarthy MI, Hattersley AT, Frayling TM 2003 Large-scale association studies of variants in genes encoding the pancreatic ß-cell KATP channel subunits Kir6.2 (KCNJ11) and SUR1 (ABCC8) confirm that the KCNJ11 E23K variant is associated with type 2 diabetes. Diabetes 52:568–572[Abstract/Free Full Text]
  13. Bockamp E, Maringer M, Spangenberg C, Fees S, Fraser S, Eshkind L, Oesch F, Zabel B 2002 Of mice and models: improved animal models for biomedical research. Physiol Genomics 11:115–132[Abstract/Free Full Text]
  14. Morrison AD 1998 1 + 1 = r4 and much much more. Bioessays 20:794–797[CrossRef][Medline]
  15. Yla-Herttuala S, Alitalo K 2003 Gene transfer as a tool to induce therapeutic vascular growth. Nat Med 9:694–701[CrossRef][Medline]
  16. Doudna JA, Cech TR 2002 The chemical repertoire of natural ribozymes. Nature 418:222–228[CrossRef][Medline]
  17. Sullenger BA, Gilboa E 2002 Emerging clinical applications of RNA. Nature 418:252–258[CrossRef][Medline]
  18. Sczakiel G, Goody RS 1994 Antisense principle or ribozyme action? Biol Chem Hoppe Seyler 375:745–746[Medline]
  19. Hormes R, Sczakiel G 2002 The size of hammerhead ribozymes is related to cleavage kinetics: the role of substrate length. Biochimie 84:897–903[Medline]
  20. Fritz JJ, Lewin A, Hauswirth W, Agarwal A, Grant M, Shaw L 2002 Development of hammerhead ribozymes to modulate endogenous gene expression for functional studies. Methods 28:276–285[CrossRef][Medline]
  21. Irminger JC, Meyer K, Halban P 1996 Proinsulin processing in the rat insulinoma cell line INS after overexpression of the endoproteases PC2 or PC3 by recombinant adenovirus. Biochem J 320:11–15
  22. Wu J, Cui N, Piao H, Wang Y, Xu H, Mao J, Jiang C 2002 Allosteric modulation of the mouse Kir6.2 channel by intracellular H+ and ATP. J Physiol 543:495–504[Abstract/Free Full Text]
  23. Wang XR, Wu JP, Li L, Chen FX, Wang RP, Jiang C 2003 Hypercapnic acidosis activates KATP channels in vascular smooth muscles. Circ Res 92:1225–1232[Abstract/Free Full Text]
  24. Carlsson PO, Jansson L, Palm F 2002 Unaltered oxygen tension in rat pancreatic islets despite dissociation of insulin release and islet blood flow. Acta Physiol Scand 176:275–281[CrossRef][Medline]
  25. Noma A 1983 ATP-regulated K+ channels in cardiac muscle. Nature 305:147–148[CrossRef][Medline]
  26. Cook DL, Hales CN 1984 Intracellular ATP directly blocks K+ channels in pancreatic ß-cells. Nature 311:271–273[CrossRef][Medline]
  27. Ashcroft FM, Harrison DE, Ashcroft SJ 1984 Glucose induces closure of single potassium channels in isolated rat pancreatic ß-cell. Nature 312:446–448[CrossRef][Medline]
  28. Long MB, Jones JP, Sullenger BA, Byun J 2003 Ribozyme-mediated revision of RNA and DNA. J Clin Invest 112:312–318[CrossRef][Medline]
  29. Gibson SA, Pellenz C, Hutchison RE, Davey FR, Shillitoe EJ 2000 Induction of apoptosis in oral cancer cells by an anti-bcl-2 ribozyme delivered by an adenovirus vector. Clin Cancer Res 6:213–222[Abstract/Free Full Text]
  30. Jarvis TC, Bouhana KS, Lesch ME, Brown SA, Parry TJ, Schrier DJ, Hunt SW, Pavco PA, Flory CM 2000 Ribozymes as tools for therapeutic target validation in arthritis. J Immunol 165:493–498[Abstract/Free Full Text]
  31. Murray JB, Seyhan AA, Walter NG, Burke JM, Scott WG 1998 The hammerhead, hairpin and VS ribozymes are catalytically proficient in monovalent cations alone. Chem Biol 5:587–595[CrossRef][Medline]
  32. Salehi-Ashtiani K, Szostak JW 2001 In vitro evolution suggests multiple origins for the hammerhead ribozyme. Nature 414:82–84[CrossRef][Medline]
  33. Tucker SJ, Gribble FM, Proks P, Trapp S, Ryder TJ, Haug T, Reimann F, Ashcroft FM 1998 Molecular determinants of KATP channel inhibition by ATP. EMBO J 17:3290–3296[CrossRef][Medline]
  34. Drain P, Li L, Wang J 1998 KATP channel inhibition by ATP requires distinct functional domains of the cytoplasmic C terminus of the pore-forming subunit. Proc Natl Acad Sci USA 95:13953–13958[Abstract/Free Full Text]
  35. Piao H, Cui N, Xu H, Mao J, Rojas A, Wang R, Abdulkadir L, Li L, Wu J, Jiang C 2001 Requirement of multiple protein domains and residues for gating KATP channels by intracellular pH. J Biol Chem 276:36673–36680[Abstract/Free Full Text]
  36. Klein RL, McNamara RK, King MA, Lenox RH, Muzyczka N, Meyer EM 1999 Generation of aberrant sprouting in the adult rat brain by GAP-43 somatic gene transfer. Brain Res 832:136–144[CrossRef][Medline]
  37. DelloRusso C, Scott J, Hartigan-O’Connor DJ, Robinson A, Brooks SV, Chamberlain JS 1999 Functional correction of old mdxmouse muscle using a gutted adenoviral vector expressing human dystrophin. Am J Hum Genet 65:A25
  38. Schiedner G, Hertel S, Johnston M, Dries V, van Rooijen N, Kochanek S 2003 Selective depletion or blockade of Kupffer cells leads to enhanced and prolonged hepatic transgene expression using high-capacity adenoviral vectors. Mol Ther 7:35–43[CrossRef][Medline]
  39. Kashani-Sabet M, Liu Y, Fong S, Desprez PY, Liu SQ, Tu GH, Nosrati M, Handumrongkul C, Liggitt D, Thor AD, Debs RJ 2002 Identification of gene function and functional pathways by systemic plasmid-based ribozyme targeting in adult mice. Proc Natl Acad Sci USA 99:3878–3883[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
X. Geng, L. Li, R. Bottino, A. N. Balamurugan, S. Bertera, E. Densmore, A. Su, Y. Chang, M. Trucco, and P. Drain
Antidiabetic sulfonylurea stimulates insulin secretion independently of plasma membrane KATP channels
Am J Physiol Endocrinol Metab, July 1, 2007; 293(1): E293 - E301.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow A correction has been published
Right arrow All Versions of this Article:
145/9/4408    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, L.
Right arrow Articles by Jiang, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, L.
Right arrow Articles by Jiang, C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals