miR-375 targets 3'-phosphoinositide-dependent protein kinase-1 and regulates glucose-induced biological responses in pancreatic beta-cells - PubMed (original) (raw)

. 2008 Oct;57(10):2708-17.

doi: 10.2337/db07-1614. Epub 2008 Jun 30.

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miR-375 targets 3'-phosphoinositide-dependent protein kinase-1 and regulates glucose-induced biological responses in pancreatic beta-cells

Abdelfattah El Ouaamari et al. Diabetes. 2008 Oct.

Abstract

Objective: MicroRNAs are short, noncoding RNAs that regulate gene expression. We hypothesized that the phosphatidylinositol 3-kinase (PI 3-kinase) cascade known to be important in beta-cell physiology could be regulated by microRNAs. Here, we focused on the pancreas-specific miR-375 as a potential regulator of its predicted target 3'-phosphoinositide-dependent protein kinase-1 (PDK1), and we analyzed its implication in the response of insulin-producing cells to elevation of glucose levels.

Research design and methods: We used insulinoma-1E cells to analyze the effects of miR-375 on PDK1 protein level and downstream signaling using Western blotting, glucose-induced insulin gene expression using quantitative RT-PCR, and DNA synthesis by measuring thymidine incorporation. Moreover, we analyzed the effect of glucose on miR-375 expression in both INS-1E cells and primary rat islets. Finally, miR-375 expression in isolated islets was analyzed in diabetic Goto-Kakizaki (GK) rats.

Results: We found that miR-375 directly targets PDK1 and reduces its protein level, resulting in decreased glucose-stimulatory action on insulin gene expression and DNA synthesis. Furthermore, glucose leads to a decrease in miR-375 precursor level and a concomitant increase in PDK1 protein. Importantly, regulation of miR-375 expression by glucose occurs in primary rat islets as well. Finally, miR-375 expression was found to be decreased in fed diabetic GK rat islets.

Conclusions: Our findings provide evidence for a role of a pancreatic-specific microRNA, miR-375, in the regulation of PDK1, a key molecule in PI 3-kinase signaling in pancreatic beta-cells. The effects of glucose on miR-375 are compatible with the idea that miR-375 is involved in glucose regulation of insulin gene expression and beta-cell growth.

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Figures

FIG. 1.

FIG. 1.

PDK1 is a target of miR-375. A: Scheme of the interaction between miR-375 and the 3′UTR of mouse, rat, and human PDK1. B: Quantification of miR-375 precursor levels. INS-1E cells were transfected with pNeg or pmiR-375 for 48 h. miR-375 precursor was then quantified by quantitative RT-PCR. Values for miR-375 precursor were normalized to U6 RNA. C: Analysis of PDK1 protein. INS-1E cells were transfected as above, and protein extracts were analyzed by Western blot using antibody to PDK1 or to β-tubulin. D: Relative quantification of PDK1 protein. Data represent three independent transfections done in triplicate, ±SE, with n = 3. *P < 0.05. E: Relative quantification of PDK1 mRNA levels. RNA extracts were used for quantitative RT-PCR analysis of PDK1 mRNA normalized to 36B4 mRNA. Data represent three independent transfections done in triplicate, ±SE, with n = 3. F: Study of the interaction between miR-375 and 3′UTR of PDK1 mRNA. INS-1E cells were cotransfected with one of the following pmiR-reporter luciferase vectors (Ambion): empty vector, vector containing the 3′UTR PDK-1 oligonucleotide predicted to interact with miR-375, or vector containing a mutated sequence. The cells were also transfected with either pmiR-375 or pNeg. Forty-eight hours after transfection, cells were assayed for luciferase and β-galactosidase activity; β-galactodidase was used as control to normalize for transfection efficiency. Data represent three independent transfections, each carried out in duplicate, ±SE, with n = 3. *P < 0.05.

FIG. 2.

FIG. 2.

Effect of miR-375 on PKB and GSK3 phosphorylation. A: Analysis of Thr-308-phosphorylation of PKB. INS-1E cells were transfected with pNeg or pmiR-375. Forty-eight hours later, cells were starved in Krebs-Ringer bicarbonate HEPES medium for 2 h and either were or were not stimulated with 100 nmol/l insulin for 5 min. Protein extracts were analyzed by Western blot using antibody to phospho308Thr PKB and total PKB. B: Quantification of Thr-308-phosphorylated PKB. C: Analysis of GSK3β phosphorylation. Protein extracts were analyzed by Western blot using antibody to phosphoGSK3β and total GSK3. D: Quantification of phosphorylated GSK3β. The data presented correspond to three independent experiments, each done in duplicate, ±SE, with n = 3. *P < 0.05.

FIG. 3.

FIG. 3.

Effect of miR-375 on glucose-enhanced insulin gene expression and cell proliferation. A: Quantification of insulin mRNA. INS-1E cells were transfected as above. Forty-eight hours later, cells were starved in RPMI 1640 with 0.5% (vol/vol) FCS containing 2 mmol/l glucose for 16 h and treated with 2 or 20 mmol/l glucose for 1 h. Insulin mRNA expression was analyzed by quantitative RT-PCR and normalized to 36B4 transcript. Data represent five independent experiments carried out in triplicate, ±SE, with n = 5. *P < 0.05, **P < 0.005. B: Measurement of [methyl-3H]thymidine incorporation. INS-1E cells were transfected as above. Twenty-four hours later, cells were starved in RPMI 1640 with 0.5% (vol/vol) FCS containing 2 mmol/l glucose for 16 h and treated with 2 or 20 mmol/l glucose for 24 h, and cell proliferation was assessed by measuring [methyl-3H]thymidine incorporation. Data represent three independent experiments done in triplicate, ±SE, with n = 3. *P < 0.05.

FIG. 4.

FIG. 4.

Effect of miR-375 on cell number, viability, and proliferation. A: Cell number counting. INS-1E cells were seeded in six-well plates (5 × 105/well) and transfected with pNeg or premiR-375. Cells were starved in RPMI 1640 containing 0.5% (vol/vol) FCS and 11 mmol/l glucose for 24 h and then replaced in RPMI 1640 10% (vol/vol) FCS and 11 mmol/l glucose for 24 h. Cells were counted using Coulter counter. B: Cell viability assay. INS-1E cells were seeded in 12-well plates (2 × 105/well) and transfected as above. Forty-eight hours later, cell viability was assessed as described in

research design and methods

. Data represent three independent transfections done in sextuplicate, ±SE, with n = 3. *P < 0.05. C: Measurement of [methyl-3H]thymidine incorporation. Cells were seeded in six-well plates (5 × 105/well) and treated as in A. Cell proliferation was assessed by measuring [methyl-3H]thymidine incorporation. Data represent three independent experiments, each run in triplicate, ±SE, with n = 3. *P < 0.05.

FIG. 5.

FIG. 5.

Effect of 2′_-O-_methyl-miR-375 antisense oligonucleotides on PDK1, glucose-enhanced insulin mRNA, and cell proliferation. A: Analysis of PDK1 protein. INS-1E cells were transfected with indicated amounts of 2′_-O-_methyl-miR-375. After 48 h, protein extracts were analyzed by Western blot using antibody to PDK1 or to β-tubulin. B: Quantification of PDK1 protein. Data represent three independent transfections, ±SE, with n = 3. **P < 0.01. C: Quantification of insulin mRNA level. INS-1E cells were transfected with either 2′_-O-_methyl-GFP or 2′_-O-_methyl-miR-375 at 500 nmol/l. Twenty-four hours later, cells were starved in RPMI 1640 with 0.5% (vol/vol) FCS containing 2 mmol/l glucose for 16 h and treated with 2 or 20 mmol/l glucose for 1 h. RNA extracts were reverse-transcribed and analyzed by RT-PCR for the expression of insulin gene normalized to the 36B4 transcript level. Data represent three independent experiments done in triplicate, ±SE, with n = 3. *P < 0.05, **P < 0.01. D: Measurement of [methyl-3H]thymidine incorporation. INS-1E cells were transfected as described above. Twenty-four hours later, cells were starved in RPMI 1640 with 0.5% (vol/vol) FCS containing 2 mmol/l glucose for 16 h and treated with 2 or 20 mmol/l glucose for 24 h, and [methyl-3H]thymidine incorporation was measured. Data represent four independent experiments, done in triplicate, ±SE, with n = 4. *P < 0.05, ***P < 0.001.

FIG. 6.

FIG. 6.

Expression of microRNAs in glucose-stimulated INS-1E cells. INS-1E cells were cultured in six-well plates (5 × 105/well) and starved in RPMI 1640 with 0.5% (vol/vol) FCS containing 2 mmol/l glucose for 24 h and thereafter treated with 5.5, 11, or 22 mmol/l glucose for 4 days. RNA extracts were reverse-transcribed and analyzed by RT-PCR for the expression of miR-375 (A), miR-296 (B), miR-9 (C), and miR-122 (D) precursors. Expression levels of microRNA precursors were normalized to U6 transcript. Data represent three independent experiments each done in duplicate, ±SE, with n = 3. *P < 0.05, **P < 0.005. Results were expressed relative to the low glucose condition (5.5 mmol/l Glc).

FIG. 7.

FIG. 7.

Effect of glucose on endogenous miR-375 and PDK1 expression in INS-1E cells. INS-1E cells were cultured in six-well plates (5 × 105/well) and starved in RPMI 1640 with 0.5% (vol/vol) FCS containing 2 mmol/l glucose for 24 h and thereafter treated with 2 or 20 mmol/l glucose for 1 h (A) or 24 h (B). RNA extracts were analyzed for miR-375 precursor. Expression of miR-375 precursor was normalized to the U6 transcript level. Protein extracts from cells stimulated for 1 h (C) or 24 h (D) with 2 or 20 mmol/l glucose were analyzed by Western blot using antibody to PDK1 or to β-tubulin. INS-1E cells were starved in RPMI 1640 with 0.5% (vol/vol) FCS containing 2 mmol/l glucose for 16 h and treated with 2 or 20 mmol/l glucose for 1 h (E) or 24 h (F). Cell proliferation was assessed by measuring [methyl-3H]thymidine incorporation. Data represent three independent experiments done in triplicate, ±SE, with n = 3. *P < 0.05, **P < 0.005.

FIG. 8.

FIG. 8.

Study of endogenous miR-375 expression in freshly isolated rat islets. Rat islets were isolated as described in

research design and methods

. Islets were maintained overnight in Ham's F10 containing 1.8 g/l glucose supplemented with 300 mg/l glutamine, 100 mg/l streptomycin, 75 mg/l penicillin, 0.5% (wt/vol) BSA, and 2% (vol/vol) FCS. Thereafter, islets were treated with either 5, 10, or 20 mmol/l glucose for 2 h (A) or 72 h (B). RNA extracts were reverse-transcribed and analyzed by quantitative RT-PCR for miR-375 precursor. Expression of miR-375 precursor was normalized to the U6 transcript level. Results are means ± SE (n = 3/condition). *P < 0.05, **P < 0.005. Freshly isolated islets from Wistar (n = 5) and GK rats (n = 6) were prepared as previously described. RNA extracts were analyzed by quantitative RT-PCR for pre-miR-375 (C), pre-miR-124a2 (D), and insulin mRNA (E). Results are means ± SE (n = 5 for Wistar and n = 6 for GK), *P < 0.05.

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