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

El Ouaamari A, Baroukh N, Martens GA, Lebrun P, Pipeleers D, van Obberghen E - Diabetes (2008)

Bottom Line: 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.Finally, miR-375 expression was found to be decreased in fed diabetic GK rat islets.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.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale, U907, Nice, France.

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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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.
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f5: 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.

Mentions: Because miR-375 targets PDK1 and impairs glucose-stimulated insulin gene expression and cell proliferation, we investigated whether antisense oligonucleotides of miR-375 induce effects opposite to those seen after miR-375 overexpression. Using 2′-O-methyl-miR-375 antisense oligonucleotides, we found that blocking miR-375 augments PDK1 protein. This increase reaches ∼40% when cells are transfected with either 250 or 500 nmol/l 2′-O-methyl-miR-375 (Fig. 5A and B). Importantly, we found that 2′-O-methyl-miR-375–induced miR-375 depletion increases both basal and glucose-enhanced insulin mRNA (Fig. 5C). Finally, as shown in (Fig. 5D), 2′-O-methyl-miR-375 increases the glucose stimulatory action on [3H]thymidine incorporation compared with 2′-O-methyl-GFP.


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

El Ouaamari A, Baroukh N, Martens GA, Lebrun P, Pipeleers D, van Obberghen E - Diabetes (2008)

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.
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f5: 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.
Mentions: Because miR-375 targets PDK1 and impairs glucose-stimulated insulin gene expression and cell proliferation, we investigated whether antisense oligonucleotides of miR-375 induce effects opposite to those seen after miR-375 overexpression. Using 2′-O-methyl-miR-375 antisense oligonucleotides, we found that blocking miR-375 augments PDK1 protein. This increase reaches ∼40% when cells are transfected with either 250 or 500 nmol/l 2′-O-methyl-miR-375 (Fig. 5A and B). Importantly, we found that 2′-O-methyl-miR-375–induced miR-375 depletion increases both basal and glucose-enhanced insulin mRNA (Fig. 5C). Finally, as shown in (Fig. 5D), 2′-O-methyl-miR-375 increases the glucose stimulatory action on [3H]thymidine incorporation compared with 2′-O-methyl-GFP.

Bottom Line: 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.Finally, miR-375 expression was found to be decreased in fed diabetic GK rat islets.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.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale, U907, Nice, France.

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.

Show MeSH