Limits...
High glucose suppresses human islet insulin biosynthesis by inducing miR-133a leading to decreased polypyrimidine tract binding protein-expression.

Fred RG, Bang-Berthelsen CH, Mandrup-Poulsen T, Grunnet LG, Welsh N - PLoS ONE (2010)

Bottom Line: Culture in high glucose resulted in increased islet contents of miR-133a and reduced contents of miR-146.However, both PTB protein levels and insulin biosynthesis rates were decreased in response to high glucose.The miR-133a inhibitor prevented the high glucose-induced decrease in PTB and insulin biosynthesis, and the miR-133a precursor decreased PTB levels and insulin biosynthesis similarly to high glucose.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden.

ABSTRACT

Background: Prolonged periods of high glucose exposure results in human islet dysfunction in vitro. The underlying mechanisms behind this effect of high glucose are, however, unknown. The polypyrimidine tract binding protein (PTB) is required for stabilization of insulin mRNA and the PTB mRNA 3'-UTR contains binding sites for the microRNA molecules miR-133a, miR-124a and miR-146. The aim of this study was therefore to investigate whether high glucose increased the levels of these three miRNAs in association with lower PTB levels and lower insulin biosynthesis rates.

Methodology/principal findings: Human islets were cultured for 24 hours in the presence of low (5.6 mM) or high glucose (20 mM). Islets were also exposed to sodium palmitate or the proinflammatory cytokines IL-1beta and IFN-gamma, since saturated free fatty acids and cytokines also cause islet dysfunction. RNA was then isolated for real-time RT-PCR analysis of miR-133a, miR-124a, miR-146, insulin mRNA and PTB mRNA contents. Insulin biosynthesis rates were determined by radioactive labeling and immunoprecipitation. Synthetic miR-133a precursor and inhibitor were delivered to dispersed islet cells by lipofection, and PTB was analyzed by immunoblotting following culture at low or high glucose. Culture in high glucose resulted in increased islet contents of miR-133a and reduced contents of miR-146. Cytokines increased the contents of miR-146. The insulin and PTB mRNA contents were unaffected by high glucose. However, both PTB protein levels and insulin biosynthesis rates were decreased in response to high glucose. The miR-133a inhibitor prevented the high glucose-induced decrease in PTB and insulin biosynthesis, and the miR-133a precursor decreased PTB levels and insulin biosynthesis similarly to high glucose.

Conclusion: Prolonged high-glucose exposure down-regulates PTB levels and insulin biosynthesis rates in human islets by increasing miR-133a levels. We propose that this mechanism contributes to hyperglycemia-induced beta-cell dysfunction.

Show MeSH

Related in: MedlinePlus

Effects of high glucose, palmitate or cytokines on human islet PTB protein levels.Human islets were cultured as given in Figure 1. Islets were washed and harvested for SDS-PAGE and immunoblotting with anti-PTB antibodies. PTB bands were quantified and normalized to total protein loading using Amidoblack staining. Lower panel shows PTB immunoreactivity and Amidoblack staining of total protein loading from one representative experiment. Results are means ± SEM for 4 independent experiments. * denotes p<0.05 using paired Student's t-test when comparing vs. control.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2877094&req=5

pone-0010843-g003: Effects of high glucose, palmitate or cytokines on human islet PTB protein levels.Human islets were cultured as given in Figure 1. Islets were washed and harvested for SDS-PAGE and immunoblotting with anti-PTB antibodies. PTB bands were quantified and normalized to total protein loading using Amidoblack staining. Lower panel shows PTB immunoreactivity and Amidoblack staining of total protein loading from one representative experiment. Results are means ± SEM for 4 independent experiments. * denotes p<0.05 using paired Student's t-test when comparing vs. control.

Mentions: Having observed that a deleterious concentration of glucose and cytokines increased miR-133a and miR-146, respectively, we next investigated whether PTB mRNA and PTB protein levels were affected. The thresh-hold cycle numbers for PTB1 were 3–4 cycles lower (29 vs. 33 cycles) than those for PTB2, indicating strong expression of PTB1 in human islet cells. None of the experimental conditions altered PTB1- or PTB2 mRNA levels (Figure 2A and 2B). However, PTB protein levels were decreased by 30–40% in islets incubated in the presence of high glucose or sodium palmitate (Figure 3) consistent with the observation that miR-133a inhibits PTB mRNA translation by binding to the 3′-UTR of PTB mRNA [31], [32], and in line with the generally accepted notion that miRNAs often decrease translation without affecting mRNA stability and levels [38], [39]. No effect was observed in response to cytokines (Figure 3), indicating that miR-146, which was strongly induced by cytokines (Figure 1C), does not target PTB-mRNA in human islet cells.


High glucose suppresses human islet insulin biosynthesis by inducing miR-133a leading to decreased polypyrimidine tract binding protein-expression.

Fred RG, Bang-Berthelsen CH, Mandrup-Poulsen T, Grunnet LG, Welsh N - PLoS ONE (2010)

Effects of high glucose, palmitate or cytokines on human islet PTB protein levels.Human islets were cultured as given in Figure 1. Islets were washed and harvested for SDS-PAGE and immunoblotting with anti-PTB antibodies. PTB bands were quantified and normalized to total protein loading using Amidoblack staining. Lower panel shows PTB immunoreactivity and Amidoblack staining of total protein loading from one representative experiment. Results are means ± SEM for 4 independent experiments. * denotes p<0.05 using paired Student's t-test when comparing vs. control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2877094&req=5

pone-0010843-g003: Effects of high glucose, palmitate or cytokines on human islet PTB protein levels.Human islets were cultured as given in Figure 1. Islets were washed and harvested for SDS-PAGE and immunoblotting with anti-PTB antibodies. PTB bands were quantified and normalized to total protein loading using Amidoblack staining. Lower panel shows PTB immunoreactivity and Amidoblack staining of total protein loading from one representative experiment. Results are means ± SEM for 4 independent experiments. * denotes p<0.05 using paired Student's t-test when comparing vs. control.
Mentions: Having observed that a deleterious concentration of glucose and cytokines increased miR-133a and miR-146, respectively, we next investigated whether PTB mRNA and PTB protein levels were affected. The thresh-hold cycle numbers for PTB1 were 3–4 cycles lower (29 vs. 33 cycles) than those for PTB2, indicating strong expression of PTB1 in human islet cells. None of the experimental conditions altered PTB1- or PTB2 mRNA levels (Figure 2A and 2B). However, PTB protein levels were decreased by 30–40% in islets incubated in the presence of high glucose or sodium palmitate (Figure 3) consistent with the observation that miR-133a inhibits PTB mRNA translation by binding to the 3′-UTR of PTB mRNA [31], [32], and in line with the generally accepted notion that miRNAs often decrease translation without affecting mRNA stability and levels [38], [39]. No effect was observed in response to cytokines (Figure 3), indicating that miR-146, which was strongly induced by cytokines (Figure 1C), does not target PTB-mRNA in human islet cells.

Bottom Line: Culture in high glucose resulted in increased islet contents of miR-133a and reduced contents of miR-146.However, both PTB protein levels and insulin biosynthesis rates were decreased in response to high glucose.The miR-133a inhibitor prevented the high glucose-induced decrease in PTB and insulin biosynthesis, and the miR-133a precursor decreased PTB levels and insulin biosynthesis similarly to high glucose.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden.

ABSTRACT

Background: Prolonged periods of high glucose exposure results in human islet dysfunction in vitro. The underlying mechanisms behind this effect of high glucose are, however, unknown. The polypyrimidine tract binding protein (PTB) is required for stabilization of insulin mRNA and the PTB mRNA 3'-UTR contains binding sites for the microRNA molecules miR-133a, miR-124a and miR-146. The aim of this study was therefore to investigate whether high glucose increased the levels of these three miRNAs in association with lower PTB levels and lower insulin biosynthesis rates.

Methodology/principal findings: Human islets were cultured for 24 hours in the presence of low (5.6 mM) or high glucose (20 mM). Islets were also exposed to sodium palmitate or the proinflammatory cytokines IL-1beta and IFN-gamma, since saturated free fatty acids and cytokines also cause islet dysfunction. RNA was then isolated for real-time RT-PCR analysis of miR-133a, miR-124a, miR-146, insulin mRNA and PTB mRNA contents. Insulin biosynthesis rates were determined by radioactive labeling and immunoprecipitation. Synthetic miR-133a precursor and inhibitor were delivered to dispersed islet cells by lipofection, and PTB was analyzed by immunoblotting following culture at low or high glucose. Culture in high glucose resulted in increased islet contents of miR-133a and reduced contents of miR-146. Cytokines increased the contents of miR-146. The insulin and PTB mRNA contents were unaffected by high glucose. However, both PTB protein levels and insulin biosynthesis rates were decreased in response to high glucose. The miR-133a inhibitor prevented the high glucose-induced decrease in PTB and insulin biosynthesis, and the miR-133a precursor decreased PTB levels and insulin biosynthesis similarly to high glucose.

Conclusion: Prolonged high-glucose exposure down-regulates PTB levels and insulin biosynthesis rates in human islets by increasing miR-133a levels. We propose that this mechanism contributes to hyperglycemia-induced beta-cell dysfunction.

Show MeSH
Related in: MedlinePlus