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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 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.
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f2: 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.

Mentions: The PI 3-kinase/PDK1/PKB signaling pathway is used by insulin in pancreatic β-cells to elicit several actions of the hormone. It is generally believed that after insulin stimulation of cells, PDK1 is recruited to the plasma membrane and phosphorylates PKB on Thr-308, which becomes activated and phosphorylates a series of substrates, including GSK3α/β. Downregulation of PDK1 in β-cells is expected to cause a decrease in insulin-induced signaling dependent on this particular enzyme. To study the effect of miR-375 on insulin signaling, we examined the phosphorylation state of molecules functioning downstream of PDK1. Immunoblot analysis showed that in response to insulin, PKB phosphorylation on Thr-308 is less abundant in cells overexpressing miR-375 precursor compared with control cells (Fig. 2A and B). Consistent with this latter observation, ectopic expression of miR-375 precursor also reduces insulin-induced phosphorylation of GSK3β (Fig. 2C and D).


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

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

f2: 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.
Mentions: The PI 3-kinase/PDK1/PKB signaling pathway is used by insulin in pancreatic β-cells to elicit several actions of the hormone. It is generally believed that after insulin stimulation of cells, PDK1 is recruited to the plasma membrane and phosphorylates PKB on Thr-308, which becomes activated and phosphorylates a series of substrates, including GSK3α/β. Downregulation of PDK1 in β-cells is expected to cause a decrease in insulin-induced signaling dependent on this particular enzyme. To study the effect of miR-375 on insulin signaling, we examined the phosphorylation state of molecules functioning downstream of PDK1. Immunoblot analysis showed that in response to insulin, PKB phosphorylation on Thr-308 is less abundant in cells overexpressing miR-375 precursor compared with control cells (Fig. 2A and B). Consistent with this latter observation, ectopic expression of miR-375 precursor also reduces insulin-induced phosphorylation of GSK3β (Fig. 2C and D).

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