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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

Effect of palmitate on miR34a and miR146 expression. MIN6B1 cells (left) were cultured for 72 h in normal DMEM (25 mmol/l glucose concentration; Control) or in DMEM supplemented with 1 mmol/l palmitate (Pal). Freshly isolated rat pancreatic islets (right) were cultured for 72 h in normal RPMI 1640 (11 mmol/l glucose concentration; Control), in RPMI 1640 containing 25 mmol/l glucose (Glc), in RPMI 1640 containing 1 mmol/l palmitate (Pal), or in RPMI 1640 containing 25 mmol/l glucose and 1 mmol/l palmitate (Glc/Pal). The expression of miR34a and miR146 was assessed by quantitative RT-PCR. The level of U6 measured in parallel in the same samples was used to normalize the data. The asterisks indicate the conditions significantly different (P < 0.05) from control.
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f1: Effect of palmitate on miR34a and miR146 expression. MIN6B1 cells (left) were cultured for 72 h in normal DMEM (25 mmol/l glucose concentration; Control) or in DMEM supplemented with 1 mmol/l palmitate (Pal). Freshly isolated rat pancreatic islets (right) were cultured for 72 h in normal RPMI 1640 (11 mmol/l glucose concentration; Control), in RPMI 1640 containing 25 mmol/l glucose (Glc), in RPMI 1640 containing 1 mmol/l palmitate (Pal), or in RPMI 1640 containing 25 mmol/l glucose and 1 mmol/l palmitate (Glc/Pal). The expression of miR34a and miR146 was assessed by quantitative RT-PCR. The level of U6 measured in parallel in the same samples was used to normalize the data. The asterisks indicate the conditions significantly different (P < 0.05) from control.

Mentions: Prolonged exposure to FFAs has deleterious impacts on pancreatic β-cell function, including alterations in insulin secretion and sensitization toward apoptosis (2,3). Three-day exposure to palmitate resulted in analogous defects in the well-differentiated mouse insulin-secreting cell line MIN6B1 (12) (see below). miRNAs are newly discovered gene regulators that have been shown to control insulin secretion (7,9–11). These noncoding RNAs have also been demonstrated to regulate apoptosis in different cell systems (21). We therefore investigated whether changes in the level of miRNAs can contribute to the effects of FFAs. For this purpose, we initially compared the global miRNA expression profile of MIN6B1 cells cultured in the presence or absence of 1 mmol/l palmitate for 3 days. Microarray analysis permitted the detection of 132 miRNAs expressed in MIN6B1 cells (supplemental Table 1, which is available in an online appendix at http://dx.doi.org 10.2337/db07-1252). With prolonged incubation with palmitate, we observed no significant changes in the level of most miRNAs, including miR375, miR9, and miR124a, the three miRNAs previously involved in the regulation of pancreatic β-cell functions (7,9–11) (supplemental Table 1). However, after chronic exposure to palmitate, the level of a small group of miRNAs appeared to be altered. The expression of this subset of miRNAs, including miR34a, miR96, miR145, miR146, miR195, and miR210, was analyzed in more detail by quantitative RT-PCR in a large series of samples obtained from MIN6B1 cells and freshly isolated rat pancreatic islets. The relatively small variations in the levels of miR96, miR145, and miR195 detected in the microarray profiling could not be replicated in all samples (supplementary Fig. 1), whereas the decrease in miR210 expression was confirmed in MIN6B1 cells but could not be reproduced in rat pancreatic islets (supplementary Fig. 2). In view of these findings, these miRNAs were not further analyzed, and we focused on miR34a and miR146, which are increased in MIN6B1 cells and in rat pancreatic islets (Fig. 1). Exposure of pancreatic islets to elevated glucose concentrations have been shown to exacerbate the effect of FFAs (22). Incubations of rat pancreatic islets in the presence of 25 mmol/l glucose resulted in a small increase of miR34a but did not potentiate the effect of palmitate (Fig. 1). The presence of supraphysiological concentrations of glucose did not modify the expression of miR146. The relevance of our in vitro observations was verified by measuring the expression of these two miRNAs in pancreatic islets from a mouse model of type 2 diabetes. Elevated plasma FFA concentrations, β-cell dysfunction, and diabetes have been displayed in 14- to 20-week-old db/db obese mice (14). We found that in pancreatic islets from diabetic db/db mice, the levels of miR34a and miR146 were significantly higher compared with age-matched wild-type mice (Fig. 2), whereas the level of miR124a was unchanged (not shown).


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)

Effect of palmitate on miR34a and miR146 expression. MIN6B1 cells (left) were cultured for 72 h in normal DMEM (25 mmol/l glucose concentration; Control) or in DMEM supplemented with 1 mmol/l palmitate (Pal). Freshly isolated rat pancreatic islets (right) were cultured for 72 h in normal RPMI 1640 (11 mmol/l glucose concentration; Control), in RPMI 1640 containing 25 mmol/l glucose (Glc), in RPMI 1640 containing 1 mmol/l palmitate (Pal), or in RPMI 1640 containing 25 mmol/l glucose and 1 mmol/l palmitate (Glc/Pal). The expression of miR34a and miR146 was assessed by quantitative RT-PCR. The level of U6 measured in parallel in the same samples was used to normalize the data. The asterisks indicate the conditions significantly different (P < 0.05) from control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Effect of palmitate on miR34a and miR146 expression. MIN6B1 cells (left) were cultured for 72 h in normal DMEM (25 mmol/l glucose concentration; Control) or in DMEM supplemented with 1 mmol/l palmitate (Pal). Freshly isolated rat pancreatic islets (right) were cultured for 72 h in normal RPMI 1640 (11 mmol/l glucose concentration; Control), in RPMI 1640 containing 25 mmol/l glucose (Glc), in RPMI 1640 containing 1 mmol/l palmitate (Pal), or in RPMI 1640 containing 25 mmol/l glucose and 1 mmol/l palmitate (Glc/Pal). The expression of miR34a and miR146 was assessed by quantitative RT-PCR. The level of U6 measured in parallel in the same samples was used to normalize the data. The asterisks indicate the conditions significantly different (P < 0.05) from control.
Mentions: Prolonged exposure to FFAs has deleterious impacts on pancreatic β-cell function, including alterations in insulin secretion and sensitization toward apoptosis (2,3). Three-day exposure to palmitate resulted in analogous defects in the well-differentiated mouse insulin-secreting cell line MIN6B1 (12) (see below). miRNAs are newly discovered gene regulators that have been shown to control insulin secretion (7,9–11). These noncoding RNAs have also been demonstrated to regulate apoptosis in different cell systems (21). We therefore investigated whether changes in the level of miRNAs can contribute to the effects of FFAs. For this purpose, we initially compared the global miRNA expression profile of MIN6B1 cells cultured in the presence or absence of 1 mmol/l palmitate for 3 days. Microarray analysis permitted the detection of 132 miRNAs expressed in MIN6B1 cells (supplemental Table 1, which is available in an online appendix at http://dx.doi.org 10.2337/db07-1252). With prolonged incubation with palmitate, we observed no significant changes in the level of most miRNAs, including miR375, miR9, and miR124a, the three miRNAs previously involved in the regulation of pancreatic β-cell functions (7,9–11) (supplemental Table 1). However, after chronic exposure to palmitate, the level of a small group of miRNAs appeared to be altered. The expression of this subset of miRNAs, including miR34a, miR96, miR145, miR146, miR195, and miR210, was analyzed in more detail by quantitative RT-PCR in a large series of samples obtained from MIN6B1 cells and freshly isolated rat pancreatic islets. The relatively small variations in the levels of miR96, miR145, and miR195 detected in the microarray profiling could not be replicated in all samples (supplementary Fig. 1), whereas the decrease in miR210 expression was confirmed in MIN6B1 cells but could not be reproduced in rat pancreatic islets (supplementary Fig. 2). In view of these findings, these miRNAs were not further analyzed, and we focused on miR34a and miR146, which are increased in MIN6B1 cells and in rat pancreatic islets (Fig. 1). Exposure of pancreatic islets to elevated glucose concentrations have been shown to exacerbate the effect of FFAs (22). Incubations of rat pancreatic islets in the presence of 25 mmol/l glucose resulted in a small increase of miR34a but did not potentiate the effect of palmitate (Fig. 1). The presence of supraphysiological concentrations of glucose did not modify the expression of miR146. The relevance of our in vitro observations was verified by measuring the expression of these two miRNAs in pancreatic islets from a mouse model of type 2 diabetes. Elevated plasma FFA concentrations, β-cell dysfunction, and diabetes have been displayed in 14- to 20-week-old db/db obese mice (14). We found that in pancreatic islets from diabetic db/db mice, the levels of miR34a and miR146 were significantly higher compared with age-matched wild-type mice (Fig. 2), whereas the level of miR124a was unchanged (not shown).

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