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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

Comparison of the effects of palmitate and miR34 on the expression of components controlling insulin exocytosis. Left: MIN6B1 cells were incubated for 72 h in the absence (−) or in the presence of 1 mmol/l palmitate (+). Right: MIN6B1 cells were transfected with a control RNA duplex or with an RNA oligonucleotide corresponding to the mature form of miR34a. The expression of the indicated components of the machinery controlling insulin exocytosis was assessed by Western blotting. The figure shows representative blots. Similar results were obtained in at least three independent experiments.
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f7: Comparison of the effects of palmitate and miR34 on the expression of components controlling insulin exocytosis. Left: MIN6B1 cells were incubated for 72 h in the absence (−) or in the presence of 1 mmol/l palmitate (+). Right: MIN6B1 cells were transfected with a control RNA duplex or with an RNA oligonucleotide corresponding to the mature form of miR34a. The expression of the indicated components of the machinery controlling insulin exocytosis was assessed by Western blotting. The figure shows representative blots. Similar results were obtained in at least three independent experiments.

Mentions: Chronic exposure to palmitate leads to changes in the expression of several proteins that play an essential role in β-cell exocytosis (30–32), including VAMP2, SNAP25, Rab27A, Noc2, and Granuphilin (Fig. 7). Interestingly, VAMP2 is among the putative targets of miR34a (33,34). In agreement with the computational prediction, Western blot analysis revealed that miR34a overexpression in MIN6B1 cells results in a decrease in the level of VAMP2 (Fig. 7). In contrast, miR34 did not affect the expression of all the other components of the machinery of exocytosis tested (Fig. 7). To verify whether VAMP2 is a direct miR34a target in insulin-secreting cells, we generated a luciferase reporter construct containing the 3′UTR of VAMP2. As shown in Fig. 8A, miR34a overexpression diminished the luciferase activity produced from this construct but not from a control luciferase construct with a 3′UTR that does not contain the sequences of VAMP2 mRNA. As expected, overexpression of miR146 that has no impact on hormone secretion did not affect the activity of the control luciferase reporter or of the vector containing the 3′UTR of VAMP2 (Fig. 8A). These findings confirm that VAMP2 is a direct target of miR34a and suggest that diminished expression of this SNARE may potentially contribute to the secretory defects observed with increased expression of this miRNA.


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)

Comparison of the effects of palmitate and miR34 on the expression of components controlling insulin exocytosis. Left: MIN6B1 cells were incubated for 72 h in the absence (−) or in the presence of 1 mmol/l palmitate (+). Right: MIN6B1 cells were transfected with a control RNA duplex or with an RNA oligonucleotide corresponding to the mature form of miR34a. The expression of the indicated components of the machinery controlling insulin exocytosis was assessed by Western blotting. The figure shows representative blots. Similar results were obtained in at least three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2551683&req=5

f7: Comparison of the effects of palmitate and miR34 on the expression of components controlling insulin exocytosis. Left: MIN6B1 cells were incubated for 72 h in the absence (−) or in the presence of 1 mmol/l palmitate (+). Right: MIN6B1 cells were transfected with a control RNA duplex or with an RNA oligonucleotide corresponding to the mature form of miR34a. The expression of the indicated components of the machinery controlling insulin exocytosis was assessed by Western blotting. The figure shows representative blots. Similar results were obtained in at least three independent experiments.
Mentions: Chronic exposure to palmitate leads to changes in the expression of several proteins that play an essential role in β-cell exocytosis (30–32), including VAMP2, SNAP25, Rab27A, Noc2, and Granuphilin (Fig. 7). Interestingly, VAMP2 is among the putative targets of miR34a (33,34). In agreement with the computational prediction, Western blot analysis revealed that miR34a overexpression in MIN6B1 cells results in a decrease in the level of VAMP2 (Fig. 7). In contrast, miR34 did not affect the expression of all the other components of the machinery of exocytosis tested (Fig. 7). To verify whether VAMP2 is a direct miR34a target in insulin-secreting cells, we generated a luciferase reporter construct containing the 3′UTR of VAMP2. As shown in Fig. 8A, miR34a overexpression diminished the luciferase activity produced from this construct but not from a control luciferase construct with a 3′UTR that does not contain the sequences of VAMP2 mRNA. As expected, overexpression of miR146 that has no impact on hormone secretion did not affect the activity of the control luciferase reporter or of the vector containing the 3′UTR of VAMP2 (Fig. 8A). These findings confirm that VAMP2 is a direct target of miR34a and suggest that diminished expression of this SNARE may potentially contribute to the secretory defects observed with increased expression of this miRNA.

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