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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 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.
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f3: 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.

Mentions: Because PI 3-kinase signaling has been implicated in glucose-induced upregulation of insulin gene expression and because we found that miR-375 decreases PDK1 protein, we examined the impact of miR-375 on insulin gene expression in response to glucose in INS-1E cells. We found that, as expected, high glucose concentration (20 mmol/l) induced insulin gene expression in control cells, but this effect was lost in cells overexpressing pre-miR-375 (Fig. 3A). To test whether PDK1 is involved in the glucose-induced increase in cell proliferative activity, we analyzed the impact of miR-375 on cellular [methyl-3H]thymidine incorporation. As illustrated in Fig. 3B, the stimulatory effect of glucose on DNA synthesis was reduced by ∼50% when miR-375 precursor was overexpressed.


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 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2551681&req=5

f3: 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.
Mentions: Because PI 3-kinase signaling has been implicated in glucose-induced upregulation of insulin gene expression and because we found that miR-375 decreases PDK1 protein, we examined the impact of miR-375 on insulin gene expression in response to glucose in INS-1E cells. We found that, as expected, high glucose concentration (20 mmol/l) induced insulin gene expression in control cells, but this effect was lost in cells overexpressing pre-miR-375 (Fig. 3A). To test whether PDK1 is involved in the glucose-induced increase in cell proliferative activity, we analyzed the impact of miR-375 on cellular [methyl-3H]thymidine incorporation. As illustrated in Fig. 3B, the stimulatory effect of glucose on DNA synthesis was reduced by ∼50% when miR-375 precursor was overexpressed.

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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