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Alterations in microRNA expression contribute to fatty acid-induced pancreatic beta-cell dysfunction.

Lovis P, Roggli E, Laybutt DR, Gattesco S, Yang JY, Widmann C, Abderrahmani A, Regazzi R - Diabetes (2008)

Bottom Line: The latter effect is associated with inhibition of the expression of vesicle-associated membrane protein 2, a key player in beta-cell exocytosis.Higher miR146 levels do not affect the capacity to release insulin but contribute to increased apoptosis.Our findings suggest that at least part of the detrimental effects of palmitate on beta-cells is caused by alterations in the level of specific miRNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Morphology, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland.

ABSTRACT

Objective: Visceral obesity and elevated plasma free fatty acids are predisposing factors for type 2 diabetes. Chronic exposure to these lipids is detrimental for pancreatic beta-cells, resulting in reduced insulin content, defective insulin secretion, and apoptosis. We investigated the involvement in this phenomenon of microRNAs (miRNAs), a class of noncoding RNAs regulating gene expression by sequence-specific inhibition of mRNA translation.

Research design and methods: We analyzed miRNA expression in insulin-secreting cell lines or pancreatic islets exposed to palmitate for 3 days and in islets from diabetic db/db mice. We studied the signaling pathways triggering the changes in miRNA expression and determined the impact of the miRNAs affected by palmitate on insulin secretion and apoptosis.

Results: Prolonged exposure of the beta-cell line MIN6B1 and pancreatic islets to palmitate causes a time- and dose-dependent increase of miR34a and miR146. Elevated levels of these miRNAs are also observed in islets of diabetic db/db mice. miR34a rise is linked to activation of p53 and results in sensitization to apoptosis and impaired nutrient-induced secretion. The latter effect is associated with inhibition of the expression of vesicle-associated membrane protein 2, a key player in beta-cell exocytosis. Higher miR146 levels do not affect the capacity to release insulin but contribute to increased apoptosis. Treatment with oligonucleotides that block miR34a or miR146 activity partially protects palmitate-treated cells from apoptosis but is insufficient to restore normal secretion.

Conclusions: Our findings suggest that at least part of the detrimental effects of palmitate on beta-cells is caused by alterations in the level of specific miRNAs.

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Related in: MedlinePlus

Identification of the signaling pathway leading to the induction of miR34a. A: MIN6B1 cells were transfected with luciferase reporter plasmids driven by the wild-type miR34a promoter (Promo miR34a), by a miR34a promoter lacking the p53 binding site (Promo mut miR34a), or by p53-responsive elements (p53 Sensor). Each of these luciferase reporters was cotransfected with an empty vector (control) or with a p53-overexpressing plasmid (p53). Luciferase activities were measured 3 days later and normalized to controls. *Conditions significantly different (P < 0.05, n = 3) from controls. B: MIN6B1 cells were transfected with an empty vector or with a plasmid encoding p53. The expression of miR34a was measured by quantitative RT-PCR 2 days later. Data represent the mean of three independent experiments. *P < 0.05 (n = 3). C: MIN6B1 cells were transfected with luciferase reporter plasmids as in A and were then cultured in the absence (control) or in the presence of 1 mmol/l palmitate. Luciferase activities were measured 3 days later and normalized to controls. The results are representative of three independent experiments. *Conditions significantly different (P < 0.05) from controls. D: MIN6B1 cells (left) and isolated rat pancreatic islets (right) were cultured for 3 days in the absence (Control) or in the presence of 1 mmol/l palmitate. p53 mRNA levels were assessed by quantitative RT-PCR and normalized to the level of housekeeping gene GAPDH. Data are means ± SE of three independent experiments. *P < 0.05.
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f6: Identification of the signaling pathway leading to the induction of miR34a. A: MIN6B1 cells were transfected with luciferase reporter plasmids driven by the wild-type miR34a promoter (Promo miR34a), by a miR34a promoter lacking the p53 binding site (Promo mut miR34a), or by p53-responsive elements (p53 Sensor). Each of these luciferase reporters was cotransfected with an empty vector (control) or with a p53-overexpressing plasmid (p53). Luciferase activities were measured 3 days later and normalized to controls. *Conditions significantly different (P < 0.05, n = 3) from controls. B: MIN6B1 cells were transfected with an empty vector or with a plasmid encoding p53. The expression of miR34a was measured by quantitative RT-PCR 2 days later. Data represent the mean of three independent experiments. *P < 0.05 (n = 3). C: MIN6B1 cells were transfected with luciferase reporter plasmids as in A and were then cultured in the absence (control) or in the presence of 1 mmol/l palmitate. Luciferase activities were measured 3 days later and normalized to controls. The results are representative of three independent experiments. *Conditions significantly different (P < 0.05) from controls. D: MIN6B1 cells (left) and isolated rat pancreatic islets (right) were cultured for 3 days in the absence (Control) or in the presence of 1 mmol/l palmitate. p53 mRNA levels were assessed by quantitative RT-PCR and normalized to the level of housekeeping gene GAPDH. Data are means ± SE of three independent experiments. *P < 0.05.

Mentions: We then investigated in more detail the mechanisms responsible for the induction and action of miR34a, the miRNA with a broader impact on β-cell functions. Very recently, different independent studies have shown that the expression of miR34a is controlled by the transcription factor p53 (17,25–29). In agreement with these reports, overexpression of p53 in MIN6B1 cells increased the expression of a luciferase reporter construct driven by the miR34a promoter but not the expression of an analogous construct in which the p53 recognition site is mutated (Fig. 6A). In agreement with these findings, overexpression of p53 led to an increase in miR34a levels (Fig. 6B). We then investigated whether prolonged incubation in the presence of palmitate can activate the p53 pathway. Quantitative RT-PCR measurements revealed that under our experimental conditions, the mRNA level of p53 is increased in MIN6B1 cells and in rat pancreatic islets (Fig. 6D). In addition, we found that in cells treated with palmitate, the p53 transcriptional activity assessed using a luciferase reporter construct (p53 sensor) is enhanced (Fig. 6C), confirming the activation of the p53 pathway. As expected, prolonged incubation in the presence of palmitate augmented the activity of the miR34a (30) promoter but not the activity of the construct lacking the p53 binding site (Fig. 6C).


Alterations in microRNA expression contribute to fatty acid-induced pancreatic beta-cell dysfunction.

Lovis P, Roggli E, Laybutt DR, Gattesco S, Yang JY, Widmann C, Abderrahmani A, Regazzi R - Diabetes (2008)

Identification of the signaling pathway leading to the induction of miR34a. A: MIN6B1 cells were transfected with luciferase reporter plasmids driven by the wild-type miR34a promoter (Promo miR34a), by a miR34a promoter lacking the p53 binding site (Promo mut miR34a), or by p53-responsive elements (p53 Sensor). Each of these luciferase reporters was cotransfected with an empty vector (control) or with a p53-overexpressing plasmid (p53). Luciferase activities were measured 3 days later and normalized to controls. *Conditions significantly different (P < 0.05, n = 3) from controls. B: MIN6B1 cells were transfected with an empty vector or with a plasmid encoding p53. The expression of miR34a was measured by quantitative RT-PCR 2 days later. Data represent the mean of three independent experiments. *P < 0.05 (n = 3). C: MIN6B1 cells were transfected with luciferase reporter plasmids as in A and were then cultured in the absence (control) or in the presence of 1 mmol/l palmitate. Luciferase activities were measured 3 days later and normalized to controls. The results are representative of three independent experiments. *Conditions significantly different (P < 0.05) from controls. D: MIN6B1 cells (left) and isolated rat pancreatic islets (right) were cultured for 3 days in the absence (Control) or in the presence of 1 mmol/l palmitate. p53 mRNA levels were assessed by quantitative RT-PCR and normalized to the level of housekeeping gene GAPDH. Data are means ± SE of three independent experiments. *P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2551683&req=5

f6: Identification of the signaling pathway leading to the induction of miR34a. A: MIN6B1 cells were transfected with luciferase reporter plasmids driven by the wild-type miR34a promoter (Promo miR34a), by a miR34a promoter lacking the p53 binding site (Promo mut miR34a), or by p53-responsive elements (p53 Sensor). Each of these luciferase reporters was cotransfected with an empty vector (control) or with a p53-overexpressing plasmid (p53). Luciferase activities were measured 3 days later and normalized to controls. *Conditions significantly different (P < 0.05, n = 3) from controls. B: MIN6B1 cells were transfected with an empty vector or with a plasmid encoding p53. The expression of miR34a was measured by quantitative RT-PCR 2 days later. Data represent the mean of three independent experiments. *P < 0.05 (n = 3). C: MIN6B1 cells were transfected with luciferase reporter plasmids as in A and were then cultured in the absence (control) or in the presence of 1 mmol/l palmitate. Luciferase activities were measured 3 days later and normalized to controls. The results are representative of three independent experiments. *Conditions significantly different (P < 0.05) from controls. D: MIN6B1 cells (left) and isolated rat pancreatic islets (right) were cultured for 3 days in the absence (Control) or in the presence of 1 mmol/l palmitate. p53 mRNA levels were assessed by quantitative RT-PCR and normalized to the level of housekeeping gene GAPDH. Data are means ± SE of three independent experiments. *P < 0.05.
Mentions: We then investigated in more detail the mechanisms responsible for the induction and action of miR34a, the miRNA with a broader impact on β-cell functions. Very recently, different independent studies have shown that the expression of miR34a is controlled by the transcription factor p53 (17,25–29). In agreement with these reports, overexpression of p53 in MIN6B1 cells increased the expression of a luciferase reporter construct driven by the miR34a promoter but not the expression of an analogous construct in which the p53 recognition site is mutated (Fig. 6A). In agreement with these findings, overexpression of p53 led to an increase in miR34a levels (Fig. 6B). We then investigated whether prolonged incubation in the presence of palmitate can activate the p53 pathway. Quantitative RT-PCR measurements revealed that under our experimental conditions, the mRNA level of p53 is increased in MIN6B1 cells and in rat pancreatic islets (Fig. 6D). In addition, we found that in cells treated with palmitate, the p53 transcriptional activity assessed using a luciferase reporter construct (p53 sensor) is enhanced (Fig. 6C), confirming the activation of the p53 pathway. As expected, prolonged incubation in the presence of palmitate augmented the activity of the miR34a (30) promoter but not the activity of the construct lacking the p53 binding site (Fig. 6C).

Bottom Line: The latter effect is associated with inhibition of the expression of vesicle-associated membrane protein 2, a key player in beta-cell exocytosis.Higher miR146 levels do not affect the capacity to release insulin but contribute to increased apoptosis.Our findings suggest that at least part of the detrimental effects of palmitate on beta-cells is caused by alterations in the level of specific miRNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Morphology, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland.

ABSTRACT

Objective: Visceral obesity and elevated plasma free fatty acids are predisposing factors for type 2 diabetes. Chronic exposure to these lipids is detrimental for pancreatic beta-cells, resulting in reduced insulin content, defective insulin secretion, and apoptosis. We investigated the involvement in this phenomenon of microRNAs (miRNAs), a class of noncoding RNAs regulating gene expression by sequence-specific inhibition of mRNA translation.

Research design and methods: We analyzed miRNA expression in insulin-secreting cell lines or pancreatic islets exposed to palmitate for 3 days and in islets from diabetic db/db mice. We studied the signaling pathways triggering the changes in miRNA expression and determined the impact of the miRNAs affected by palmitate on insulin secretion and apoptosis.

Results: Prolonged exposure of the beta-cell line MIN6B1 and pancreatic islets to palmitate causes a time- and dose-dependent increase of miR34a and miR146. Elevated levels of these miRNAs are also observed in islets of diabetic db/db mice. miR34a rise is linked to activation of p53 and results in sensitization to apoptosis and impaired nutrient-induced secretion. The latter effect is associated with inhibition of the expression of vesicle-associated membrane protein 2, a key player in beta-cell exocytosis. Higher miR146 levels do not affect the capacity to release insulin but contribute to increased apoptosis. Treatment with oligonucleotides that block miR34a or miR146 activity partially protects palmitate-treated cells from apoptosis but is insufficient to restore normal secretion.

Conclusions: Our findings suggest that at least part of the detrimental effects of palmitate on beta-cells is caused by alterations in the level of specific miRNAs.

Show MeSH
Related in: MedlinePlus