The authors have declared that no competing interests exist.
Conceived and designed the experiments: EY JM. Performed the experiments: EY FT JM. Analyzed the data: EY. Contributed reagents/materials/analysis tools: EY FT JM. Wrote the paper: EY JM.
Elucidating the regulation of glucose-stimulated insulin secretion (GSIS) in pancreatic islet β cells is important for understanding and treating diabetes. MIN6 cells, a transformed β-cell line derived from a mouse insulinoma, retain GSIS and are a popular
MIN6 cells, which were isolated from an insulinoma of a transgenic mouse expressing the SV40 T antigen in pancreatic islet β cells, retain some features of differentiated pancreatic β cells
Gene expression profile analysis has been performed to compare MIN6 cells after short-term and long-term culture
In the present study, we used a MIN6 subclone, designated MIN6 clone 4, which retains GSIS even after long-term culture. To identify the genes involved in the maintenance of GSIS capacity, we compared the gene expression profiles among parental MIN6 cells after short-term culture (Pr-LP) and long-term culture (Pr-HP) and of MIN6 clone 4 cells after short-term culture (C4-LP) and long-term culture (C4-HP). The results revealed one group of genes whose expression was high in well-regulated (i.e., low passage number) parental MIN6 cells and MIN6 clone 4 cells, but extremely low in the dysregulated (high passage number) parental MIN6 cells. Another group of genes was expressed at extremely low levels in the well-regulated MIN6 cells, but at high levels in the dysregulated ones. We discuss the roles of these differentially expressed genes in insulin secretion.
MIN6 cells, which we have previously established
MIN6 cells were cultured in 24-well plates for 4 days. Prior to the insulin secretion assay, the cells were starved in Krebs Ringer solution containing 0.1% bovine serum albumin (BSA) with 3 mM glucose for 30 min, and the wells were washed twice with the same buffer. The cells were then incubated in Krebs Ringer Solution with 3, 8, 15, or 25 mM glucose, 100 µM glybenclamide+3 mM glucose, or 30 mM KCl+3 mM glucose for 1 hr.
Insulin secreted into the medium and contained in the MIN6 cells was measured. For the secreted insulin, medium was collected and the insulin measured using an ELISA kit (Cat.#10-1250-01; Mercodia, Uppsala, Sweden). To normalize the amount of secreted insulin to the protein content of each well, the cells in each well were lysed with RIPA buffer, and the protein concentration of the cell lysates was measured by the Bradford method (Cat.#500-0006; Bio-Rad, Hercules, CA). To measure the insulin content of MIN6 cells, we lysed cells in a different set of wells with acid-ethanol, and centrifuged the cell extracts. The amount of insulin in the supernatants was assayed using the ELISA kit. Statistical analysis was performed by Student′s
Total RNA was extracted from MIN6 cells using Trizol reagent (Invitrogen, Carlsbad, CA) and subjected to double-strand cDNA synthesis using the Superscript Choice system (Cat.#18090-019; Invitrogen) and the T7-(dT)24 reverse transcription primer (Cat.#72-1591-01; Amersham Biosciences, Piscataway, NJ). Synthesis of biotin-labeled cRNA was carried out by
Total RNA was extracted from MIN6 cells by the acid guanidinium-phenol-chloroform (AGPC) method and subjected to cDNA synthesis using ReverTra Ace α (Cat.#FSK-101; Toyobo, Tokyo, Japan). Quantitative RT-PCR analysis was carried out using SYBR Premix Ex Taq (Cat.#RR041A; Takara, Otsu, Japan). The reaction was performed with 1 µl cDNA per 25 µl reaction in a 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA) under the following thermal cycling conditions: 95°C for 10 sec followed by 40 cycles at 95°C for 5 sec and 60°C for 31 sec. The relative expression levels of the target genes were normalized to that of
For immunocytochemistry, the MIN6 cells were washed with PBS and fixed in 4% paraformaldehyde for 10 min, rinsed with PBS, incubated for 5 min in PBS with 1% Triton X-100, washed again with PBS, and incubated in blocking reagent (Blocking One, Cat.#03953-95; Nacalai Tesque, Kyoto, Japan). The samples were incubated with rat anti-CD24 antibody (Cat.#14-0241-81; eBioscience, San Diego, CA) for 1 hr at room temperature, washed with PBS, and then incubated with Alexa Fluor 488-conjugated anti-rat IgG (Cat.#A-11006; Molecular Probes, Eugene, OR) for 1 hr at room temperature. For FACS analysis, the cells were washed twice with PBS and then suspended in SM buffer (HEPES-buffered saline with 0.1% sodium azide and 1% BSA). CD24 staining was performed at 4°C for 30 min with rat anti-CD24 antibody, followed by incubation with Alexa Fluor 488-conjugated anti-rat IgG. FACS analyses were performed with a FACScan (Becton Dickinson, Franklin Lakes, NJ) using the CellQuest (Becton Dickinson) evaluation program.
Part of the mouse
Bisulfite treatment of the genomic DNA isolated from Pr-LP, Pr-HP, C4-LP, and C4-HP MIN6 cells was performed using the EpiTect Bisulfite Kit (Cat.#59104; Qiagen) according to the manufacturer's instructions. The CpG islands in the first intron of the
Results are presented as the mean ± SD. Statistical analyses were carried out by Student's
Insulin secretory capacity was compared by static incubation among parental MIN6 cells at 17–20 passages (Pr-LP) and 35–40 passages (Pr-HP) and MIN6 clone 4 cells at 17–20 passages (C4-LP) and 40–50 passages (C4-HP) (
Insulin secretion from Pr-LP, Pr-HP, C4-LP, and C4-HP MIN6 cells stimulated with 3 mM (3G), 8 mM (8G), 15 mM (15G), or 25 mM (25G) glucose (A, B), 100 nM glybenclamide (SU), or 30 mM KCl (C, D). Pr-HP cells showed higher basal insulin secretion at 3 mM glucose compared with Pr-LP cells, but their insulin secretion did not increase further at higher glucose concentrations or with the addition of glybenclamide or KCl, whereas both C4-LP and C4-HP MIN6 cells showed a better insulin secretory response to glucose and glybenclamide than Pr-LP cells. Values are means ± SD and n = 5–6. *
Insulin content of Pr-LP, Pr-HP, C4-LP, and C4-HP MIN6 cells (A). The insulin content of Pr-HP cells was lower than that of Pr-LP, C4-LP, or C4-HP cells. Values are means ± SD and n = 5–6. *
Furthermore, both C4-LP and C4-HP cells showed a better insulin secretory response to glucose and glybenclamide than did the Pr-LP cells (
To identify genes involved in the regulation of the insulin secretory pathway, a comparative DNA microarray analysis was performed, using the Pr-LP, Pr-HP, C4-LP, and C4-HP cells. The results revealed one group of genes, which we call “responder genes,” that were highly expressed in the responder cells (Pr-LP, C4-LP, and C4-HP), but only weakly in the non-responder cells (Pr-HP). A different group of genes (“non-responder genes”) was highly expressed in the Pr-HP cells, but only weakly in the Pr-LP, C4-LP, and C4-HP cells. To compare the responder and non-responder results, we compared the mean value of the gene expression levels in the Pr-LP, C4-LP, and C4-HP cells with the expression level in the Pr-HP cells. Genes that showed a more than 5-fold difference in expression level are listed in
Gene name | Mean value for Pr-LP, C4-LP, and C4-HP |
Pr-HP |
Fold change |
|
3306.8 | 3.8 | 876.5 |
|
750.0 | <1.0 | >750.0 |
|
2686.6 | 10.3 | 261.6 |
|
107.7 | <1.0 | >107.7 |
|
4616.1 | 114.0 | 40.5 |
|
2767.3 | 73.5 | 37.6 |
|
11245.0 | 323.7 | 34.7 |
|
119.0 | 5.4 | 22.2 |
|
1327.9 | 65.9 | 20.1 |
|
1531.1 | 78.9 | 19.4 |
|
4806.0 | 282.5 | 17.0 |
|
119.3 | 7.3 | 16.4 |
|
2463.4 | 154.3 | 16.0 |
|
798.1 | 53.2 | 15.0 |
|
210.7 | 15.2 | 13.9 |
|
607.8 | 44.8 | 13.6 |
|
7113.3 | 637.7 | 11.2 |
|
966.5 | 94.6 | 10.2 |
|
2512.2 | 250.9 | 10.0 |
|
2118.9 | 217.6 | 9.7 |
|
345.9 | 38.0 | 9.1 |
|
665.0 | 73.4 | 9.1 |
|
508.3 | 61.7 | 8.2 |
|
3705.1 | 477.8 | 7.8 |
|
252.8 | 34.9 | 7.2 |
|
2957.6 | 414.8 | 7.1 |
|
221.2 | 31.6 | 7.0 |
|
852.7 | 122.2 | 7.0 |
|
530.2 | 77.1 | 6.9 |
|
1980.3 | 288.5 | 6.9 |
|
495.8 | 73.8 | 6.7 |
|
140.7 | 21.4 | 6.6 |
|
278.1 | 42.5 | 6.6 |
|
579.0 | 93.3 | 6.2 |
|
1667.5 | 270.6 | 6.2 |
|
559.7 | 91.6 | 6.1 |
|
285.8 | 48.3 | 5.9 |
|
240.4 | 41.6 | 5.8 |
|
414.6 | 73.3 | 5.7 |
|
218.2 | 39.1 | 5.6 |
Raw values of expression intensities measured by Affymetrix arrays.
Gene name | Pr-HP |
Mean value for Pr-LP, C4-LP, and C4-HP |
Fold change |
|
2507.1 | 79.8 | 31.4 |
|
2841.2 | 93.0 | 30.6 |
|
1994.6 | 65.5 | 30.5 |
|
536.5 | 22.0 | 24.4 |
|
237.9 | 11.1 | 21.4 |
|
998.0 | 52.7 | 19.0 |
|
512.9 | 30.6 | 16.8 |
|
1035.3 | 66.6 | 15.6 |
|
2170.1 | 171.1 | 12.7 |
|
969.2 | 81.1 | 12.0 |
|
217.4 | 19.9 | 10.9 |
|
195.8 | 18.2 | 10.7 |
|
316.3 | 30.5 | 10.4 |
|
1474.6 | 160.6 | 9.2 |
|
491.0 | 54.5 | 9.0 |
|
710.5 | 86.2 | 8.2 |
|
397.9 | 50.7 | 7.9 |
|
204.5 | 26.9 | 7.6 |
|
879.6 | 117.5 | 7.5 |
|
153.9 | 21.5 | 7.2 |
|
357.1 | 52.0 | 6.9 |
|
1835.0 | 271.5 | 6.8 |
|
1365.9 | 207.3 | 6.6 |
|
4357.1 | 661.4 | 6.6 |
|
1180.7 | 188.5 | 6.3 |
|
346.5 | 55.8 | 6.2 |
|
757.0 | 123.7 | 6.1 |
|
216.5 | 35.9 | 6.0 |
|
721.4 | 120.3 | 6.0 |
|
8993.3 | 1553.4 | 5.8 |
|
634.5 | 110.2 | 5.8 |
|
566.6 | 99.7 | 5.7 |
|
603.6 | 106.8 | 5.7 |
|
1753.3 | 327.6 | 5.4 |
|
1299.8 | 243.9 | 5.3 |
|
1080.2 | 202.5 | 5.3 |
|
1234.2 | 231.4 | 5.3 |
|
984.7 | 185.9 | 5.3 |
|
669.7 | 126.7 | 5.3 |
|
199.4 | 39.5 | 5.1 |
|
379.4 | 75.8 | 5.0 |
Raw values of expression intensities measured by Affymetrix arrays.
The expression pattern of some responder and non-responder genes was examined by quantitative RT-PCR (
Expression of the
Genes encoding β-cell-related transcription factors are listed in
Gene Name | Pr-LP |
Pr-HP |
C4-LP |
C4-HP |
Mean** | Fold change*** |
|
701.4 | 519.5 | 591.7 | 570.0 | 621.0 | 0.84 |
|
2408.6 | 1395.5 | 1612.7 | 1501.1 | 1840.8 | 0.76 |
|
252.6 | 179.5 | 141.7 | 395.3 | 263.2 | 0.68 |
|
606.7 | 507.0 | 487.6 | 581.9 | 558.8 | 0.91 |
|
942.0 | 599.2 | 621.2 | 518.4 | 693.8 | 0.86 |
|
936.3 | 516.3 | 850.3 | 715.7 | 834.1 | 0.62 |
|
1896.9 | 1349.5 | 1304.2 | 1318.7 | 1506.6 | 0.90 |
|
1358.5 | 488.6 | 986.8 | 1078.4 | 1141.3 | 0.43 |
|
603.8 | 210.9 | 263.8 | 288.1 | 385.2 | 0.55 |
|
176.9 | 192.3 | 192.0 | 203.6 | 190.8 | 1.01 |
|
4580.8 | 5429.3 | 5624.6 | 3924.2 | 4709.9 | 1.15 |
|
478.5 | 499.2 | 463.8 | 1754.5 | 898.9 | 0.56 |
|
803.2 | 550.6 | 788.4 | 716.6 | 769.4 | 0.72 |
|
154.7 | 160.3 | 168.0 | 174.6 | 165.8 | 0.97 |
|
815.8 | 644.0 | 1171.2 | 933.1 | 973.4 | 0.66 |
|
28131.6 | 16852.5 | 27736.0 | 27214.7 | 27694.1 | 0.61 |
|
19939.3 | 8937.0 | 18275.3 | 20041.0 | 19418.5 | 0.46 |
|
14771.3 | 15525.9 | 15461.2 | 14919. 7 | 15050.7 | 1.03 |
|
206.1 | 206.4 | 211.3 | 191.4 | 203.0 | 1.02 |
|
810.5 | 559.8 | 524.1 | 548.7 | 627.8 | 0.89 |
|
8291.0 | 7443.6 | 9641.2 | 8110.5 | 8680.9 | 0.86 |
Raw values of expression intensities measured by Affymetrix arrays. **Mean value of C4-LP, C4-HP, and Pr-LP. ***Ratio of Pr-HP to mean value.
There was no significant difference in the expression of the insulin genes (
The DNA microarray data showed that
Immunocytochemical analysis (A) and flow cytometric analysis (B) of Pr-LP, Pr-HP, C4-LP, and C4-HP MIN6 cells with an anti-CD24 antibody. CD24 was not detected on the surface of Pr-HP cells.
Interestingly, we found several imprinted genes among the differentially expressed genes. The responder genes included
Expression of imprinted genes,
Recent reports have shown that variants in
Genomic imprinting is an epigenetic form of gene regulation that involves differential DNA methylation of the paternal and maternal alleles of a gene. Such methylation is inherited in a parent-of-origin-specific manner. We investigated the methylation status of the known regulatory regions of the
The ratio of methylated CpGs of each allele from Pr-LP, Pr-HP, C4-LP, and C4-HP MIN6 cells, in the
Although MIN6 cells are widely used as a model of pancreatic β cells, the GSIS is gradually lost with long-term culture. We previously isolated a subclone, MIN6 clone 4, from the parental MIN6 cells. This subclone showed stable GSIS even after long-term culture. To look for candidate genes for the maintenance of GSIS, we decided to analyze the gene expression patterns of Pr-LP, Pr-HP, C4-LP, and C4-HP cells. Our DNA microarray analysis extracted 60 differentially expressed responder genes (with consistently higher expression in Pr-LP, C4-LP, and C4-HP cells) and 62 non-responder genes (higher expression in the Pr-HP cells) (
Some of the responder genes have been implicated in the function or development of pancreatic β cells. One responder gene,
Genes known to be involved in exocytosis were also found among the responder genes.
The Pr-HP MIN6 cells expressed some pancreas-related genes that are not normally expressed in mature β cells. For example,
The
Our DNA microarray analysis identified
The
The present study also found a set of imprinted genes among the responder genes. The majority of known imprinted genes in mammals have roles in the control of embryonic growth and development, including development of the placenta
Two other imprinted genes,
In contrast to the above imprinted genes,
DNA methylation is necessary for the proper expression of imprinted genes
Recently, an epigenetic mechanism was reported to be involved in the dysfunction of pancreatic β cells
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The authors are grateful to Ms. Mayu Yamamoto and Mr. Masafumi Ashida for technical assistance. We acknowledge the editorial assistance of Drs. Leslie A. Miglietta and Grace E. Gray.