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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 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.
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f7: 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.

Mentions: To further document the role of miR-375, we analyzed the expression of premiR-375 in INS-1E cells treated for 1 or 24 h with 2 or 20 mmol/l glucose. We found that glucose induces a robust decrease in miR-375 after 1 and 24 h (Fig. 7A and B). Furthermore, immunoblot analyses show that PDK1 protein level is slightly, but not significantly, increased within 1 h of glucose treatment (Fig. 7C). Remarkably, in INS-1E cells exposed for 24 h to 20 mmol/l glucose, PDK1 protein is increased (fourfold), and its level thus inversely correlates with that of miR-375 (Fig. 7D). Finally, decreased miR-375 without change in PDK1 level seen after 1 h of glucose treatment occurs with enhanced insulin gene expression (Fig. 7E). Decreased miR-375 and increased PDK1 levels are associated with enhanced DNA synthesis as reflected by increased thymidine incorporation seen within 24 h of glucose treatment (Fig. 7F).


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

License
Show All Figures
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f7: 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.
Mentions: To further document the role of miR-375, we analyzed the expression of premiR-375 in INS-1E cells treated for 1 or 24 h with 2 or 20 mmol/l glucose. We found that glucose induces a robust decrease in miR-375 after 1 and 24 h (Fig. 7A and B). Furthermore, immunoblot analyses show that PDK1 protein level is slightly, but not significantly, increased within 1 h of glucose treatment (Fig. 7C). Remarkably, in INS-1E cells exposed for 24 h to 20 mmol/l glucose, PDK1 protein is increased (fourfold), and its level thus inversely correlates with that of miR-375 (Fig. 7D). Finally, decreased miR-375 without change in PDK1 level seen after 1 h of glucose treatment occurs with enhanced insulin gene expression (Fig. 7E). Decreased miR-375 and increased PDK1 levels are associated with enhanced DNA synthesis as reflected by increased thymidine incorporation seen within 24 h of glucose treatment (Fig. 7F).

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