Limits...
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.

Show MeSH

Related in: MedlinePlus

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

License
getmorefigures.php?uid=PMC2551681&req=5

f8: 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.

Mentions: To characterize the glucose-mediated miR-375 regulation, we analyzed the expression level of miR-375 precursor in isolated rat islets that had been exposed to 5, 10, or 20 mmol/l glucose for 2- or 72-h periods, which represent conditions in which the acute and chronic influences, respectively, of glucose can be studied. After 2 h at stimulatory glucose concentrations (10 or 20 mmol/l), miR-375 expression is lower than in the basal 5 mmol/l condition (Fig. 8A). After 72 h, a lower expression level was measured at 5 mmol/l than at 10 and 20 mmol/l, whereas the level at 20 mmol/l tended to be lower than at 10 mmol/l (Fig. 8B).


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)

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

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

f8: 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.
Mentions: To characterize the glucose-mediated miR-375 regulation, we analyzed the expression level of miR-375 precursor in isolated rat islets that had been exposed to 5, 10, or 20 mmol/l glucose for 2- or 72-h periods, which represent conditions in which the acute and chronic influences, respectively, of glucose can be studied. After 2 h at stimulatory glucose concentrations (10 or 20 mmol/l), miR-375 expression is lower than in the basal 5 mmol/l condition (Fig. 8A). After 72 h, a lower expression level was measured at 5 mmol/l than at 10 and 20 mmol/l, whereas the level at 20 mmol/l tended to be lower than at 10 mmol/l (Fig. 8B).

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
Related in: MedlinePlus