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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 miR34a and miR146 on hormone secretion. The mouse insulin-secreting cell line MIN6B1 was transiently transfected with a plasmid encoding hGH and with control RNA duplexes (control, palmitate); with RNA duplexes with the mature sequence of miR15b, miR34a, or minR146; or with antisense miR34a (anti-miR34a). The cells were cultured for 3 days in normal DMEM (□ and □) or with DMEM containing 1 mmol/l palmitate (▪). hGH secretion under basal conditions (top) and in the presence of stimulatory concentrations of glucose and cAMP-raising agents (bottom) was measured by ELISA. The figure shows the results of three to five independent experiments performed in triplicate. *Conditions significantly different from controls (P < 0.05).
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f4: Effect of miR34a and miR146 on hormone secretion. The mouse insulin-secreting cell line MIN6B1 was transiently transfected with a plasmid encoding hGH and with control RNA duplexes (control, palmitate); with RNA duplexes with the mature sequence of miR15b, miR34a, or minR146; or with antisense miR34a (anti-miR34a). The cells were cultured for 3 days in normal DMEM (□ and □) or with DMEM containing 1 mmol/l palmitate (▪). hGH secretion under basal conditions (top) and in the presence of stimulatory concentrations of glucose and cAMP-raising agents (bottom) was measured by ELISA. The figure shows the results of three to five independent experiments performed in triplicate. *Conditions significantly different from controls (P < 0.05).

Mentions: We first assessed the impact of the three miRNAs on the secretory properties of MIN6B1 cells. For this purpose, the cells were transfected with a plasmid leading to constitutive expression of the hGH. When introduced in β-cells, hGH is packaged in dense-core granules and is coreleased with insulin, enabling us to monitor exocytosis from transiently transfected insulin-secreting cells independently from their capacity to produce insulin (24). As shown in Fig. 4, 3-day exposure to 1 mmol/l palmitate led to the release of an exaggerated amount of hormone under basal conditions (2 mmol/l glucose) and to a reduced capacity of the cells to respond to stimulatory concentrations of 20 mmol/l glucose and cAMP-raising agents (10 μmol/l forskolin and 100 μmol/l IBMX). Overexpression of miR146 or miR15b, a miRNA whose expression is not affected by palmitate (supplemental Table 1), did not affect the secretory properties of MIN6B1 cells (Fig. 4, left). Blockade of miR146 function with an antisense oligonucleotide was also without effect (not shown). Overexpression of miR34a resulted in a significant reduction in the amount of hormone released under stimulatory conditions without modification of the secretion rate at low glucose (Fig. 4, left). Antisense miR34a treatment did not interfere with hormone secretion under basal or stimulated conditions (Fig. 4, right). Blockade of miR34a function was not sufficient to rescue a normal secretory response in cells treated with palmitate (Fig. 4, right).


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 miR34a and miR146 on hormone secretion. The mouse insulin-secreting cell line MIN6B1 was transiently transfected with a plasmid encoding hGH and with control RNA duplexes (control, palmitate); with RNA duplexes with the mature sequence of miR15b, miR34a, or minR146; or with antisense miR34a (anti-miR34a). The cells were cultured for 3 days in normal DMEM (□ and □) or with DMEM containing 1 mmol/l palmitate (▪). hGH secretion under basal conditions (top) and in the presence of stimulatory concentrations of glucose and cAMP-raising agents (bottom) was measured by ELISA. The figure shows the results of three to five independent experiments performed in triplicate. *Conditions significantly different from controls (P < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Effect of miR34a and miR146 on hormone secretion. The mouse insulin-secreting cell line MIN6B1 was transiently transfected with a plasmid encoding hGH and with control RNA duplexes (control, palmitate); with RNA duplexes with the mature sequence of miR15b, miR34a, or minR146; or with antisense miR34a (anti-miR34a). The cells were cultured for 3 days in normal DMEM (□ and □) or with DMEM containing 1 mmol/l palmitate (▪). hGH secretion under basal conditions (top) and in the presence of stimulatory concentrations of glucose and cAMP-raising agents (bottom) was measured by ELISA. The figure shows the results of three to five independent experiments performed in triplicate. *Conditions significantly different from controls (P < 0.05).
Mentions: We first assessed the impact of the three miRNAs on the secretory properties of MIN6B1 cells. For this purpose, the cells were transfected with a plasmid leading to constitutive expression of the hGH. When introduced in β-cells, hGH is packaged in dense-core granules and is coreleased with insulin, enabling us to monitor exocytosis from transiently transfected insulin-secreting cells independently from their capacity to produce insulin (24). As shown in Fig. 4, 3-day exposure to 1 mmol/l palmitate led to the release of an exaggerated amount of hormone under basal conditions (2 mmol/l glucose) and to a reduced capacity of the cells to respond to stimulatory concentrations of 20 mmol/l glucose and cAMP-raising agents (10 μmol/l forskolin and 100 μmol/l IBMX). Overexpression of miR146 or miR15b, a miRNA whose expression is not affected by palmitate (supplemental Table 1), did not affect the secretory properties of MIN6B1 cells (Fig. 4, left). Blockade of miR146 function with an antisense oligonucleotide was also without effect (not shown). Overexpression of miR34a resulted in a significant reduction in the amount of hormone released under stimulatory conditions without modification of the secretion rate at low glucose (Fig. 4, left). Antisense miR34a treatment did not interfere with hormone secretion under basal or stimulated conditions (Fig. 4, right). Blockade of miR34a function was not sufficient to rescue a normal secretory response in cells treated with palmitate (Fig. 4, right).

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