Glucose induces MafA expression in pancreatic beta cell lines via the hexosamine biosynthetic pathway

Abstract

MafA is a basic leucine zipper transcription factor that regulates gene expression in both the neuroretina and pancreas. Within the pancreas, MafA is exclusively expressed in the beta cells and is involved in insulin gene transcription, insulin secretion, and beta cell survival. The expression of the mafA gene within beta cells is known to increase in response to high glucose levels by an unknown mechanism. In this study, we demonstrate that pyruvate, which is produced by glycolysis from glucose, is not sufficient to induce mafA gene expression compared with high glucose. This suggests that the signal for MafA induction is independent of ATP levels and that a metabolic event occurring upstream of pyruvate production leads to the induction of MafA. Furthermore, insulin secretion mediated by high glucose is not important for MafA expression. However, the addition of glucosamine to beta cell lines stimulates MafA expression in the absence of high glucose, and inhibition of the hexosamine biosynthetic pathway in the presence of high glucose abolishes MafA induction. Moreover, we demonstrate that the expression of UDP-N-acetylglucosaminyl transferase, the enzyme mediating O-linked glycosylation of cytosolic and nuclear proteins, is essential for glucose-dependent MafA expression. Consistent with this observation, inhibition of N-acetylglucosaminidase, the enzyme involved in the removal of the O-GlcNAc modification from proteins, with O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate stimulates MafA expression under low glucose conditions. The presented data suggest that MafA expression mediated by high glucose requires flux through the hexosamine biosynthetic pathway and the O-linked glycosylation of an unknown protein(s) by UDP-N-acetylglucosaminyl transferase.

FIGURE 1

High glucose, but not pyruvate, induces MafA expression. MIN6 and betaTC3 cells were cultured in the presence of 20 mm pyruvate (Pyr),1mm glucose, or 25 mm glucose overnight. A, Western blot analysis of MafA and Pdx-1 protein levels. B, real time RT-PCR analysis of MafA mRNA levels. MafA mRNA levels were normalized to β-actin mRNA levels, and -fold increases were calculated based on the levels of MafA mRNA present in cells grown on 20 mm pyruvate. The data shown represent averages of two independent samples ± S.D. C, Western blot analysis of MafA protein levels in betaTC3 cells.

FIGURE 2

Hexose metabolism, but not glucose uptake, glucose phosphorylation, or osmotic stress, is required to increase MafA protein levels. A, Western blot analysis of MafA protein levels in MIN6 cells cultured with 20 or 2 mm mannose and fructose overnight. B, analysis of MafA protein levels in cells cultured overnight in the presence of 25 mm d-glucose, 30 mm 2DG, 30 mm 3-O-methyl-d-glucose (3O), or 30 mm 6-deoxy-d-glucose (6DG). C, MafA protein levels after incubation of MIN6 cells with 25 mm glucose or 30 mm l-glucose.

FIGURE 3

Induction of MafA expression by high glucose is independent of ATP levels and insulin secretion. A, Western blot analysis of MafA protein levels in MIN6 incubated with 20 mm pyruvate (Pyr) or 1, 5, 10, 15, 20, or 25 mm glucose. B, measurements of intracellular ATP levels, as described within the “Experimental Procedures,” from MIN6 cells cultured overnight in the presence of 20 mm pyruvate or 1, 5, 10, 15, 20, or 25 mm glucose. nd, not detectable. C, Western blot analysis of MafA protein levels in MIN6 cells incubated with 25 mm glucose, 1 mm glucose, 20 mm pyruvate, 50 mm KCl, or 1 μm exogenous insulin. Egr-1 was utilized as a positive control. D, analysis of glucose-, pyruvate-, and KCl-stimulated insulin secretion in MIN6 cells.

FIGURE 4

Flux through the hexosamine biosynthetic pathway is important for glucose induction of MafA expression. A, protein levels of MafA, TBP, and Pdx-1 were determined in MIN6 cells incubated with 25 mm glucose, 25 mm glucose plus 20μm DON, 1 mm glucose, or 1 mm glucose plus 2 mm GlcN. B, MafA and TBP protein levels within betaTC3 cells were analyzed by Western blotting after cells were cultured overnight in the presence of 25 mm glucose, 25 mm glucose plus 20 μm DON, or 1 mm glucose. C, Western blot analysis of MafA protein levels in MIN6 cells grown in the presence 25 mm glucose, 25 mm glucose plus 50 μm azaserine, or 1 mm glucose. D, analysis of MafA and TBP protein levels after overnight culture of MIN6 cells in the presence of 25 mm glucose, 25 mm glucose plus 20 μm DON, 25 mm glucose plus 20 μm DON plus 2 mm glucosamine, 1 mm glucose plus 2 mm glucosamine, or 1 mm glucose. E, real time RT-PCR analysis of MafA mRNA levels, normalized to β-actin mRNA levels, in MIN6 cells cultured overnight on 1 mm glucose, 1 mm glucose plus 2 mm glucosamine, 25 mm glucose, 25 mm glucose plus 20 μm DON, or 25 mm glucose plus 50 μm azaserine. -Fold increases in MafA mRNA levels are compared with mRNA levels measured in 1 mm glucose-incubated cells.

FIGURE 5

Glucose increases MafA protein levels via O-GlcNAcylation by OGT. A, Western blot analysis of MafA protein levels in MIN6 cells cultured overnight with 25 mm glucose, 1 mm glucose, or 1 mm glucose and 100 μm PUGNAc. B, OGT protein levels were knocked down within MIN6 cells using predesigned siRNA as described under “Experimental Procedures.” After OGT knockdown, MIN6 cells were cultured overnight on 25 mm glucose, after which time MafA and OGT protein levels were analyzed by Western blotting. β-Actin was used as a loading control. The blot shown is representative of two independent experiments.

FIGURE 6

Insulin mRNA levels increase in response to glucosamine. MIN6 cells were cultured in 1 mm glucose or 1 mm glucose plus 2 mm GlcN overnight. Real time RT-PCR analysis of insulin mRNA levels were normalized to β-actin mRNA levels, and -fold increases refer to insulin mRNA present in cells grown on 1 mm glucose as described under “Experimental Procedures.”

FIGURE 7

Glucose-dependent MafA expression involves the hexosamine biosynthetic pathway and O-GlcNAcylation. Flux through the HBP under high glucose conditions increases UDP-GlcNAc levels. UDP-GlcNAc is the substrate for OGT leading to the O-GlcNAcylation of an unknown protein(s) responsible for MafA up-regulation. DON and azaserine, inhibitors of GFAT, block the glucose-dependent expression of MafA, whereas GlcN treatment in the presence of low glucose stimulates MafA expression. PUGNAc, an inhibitor of O-GlcNAcase, increases MafA expression on low glucose concentrations, whereas siRNA knockdown of OGT decreases MafA expression under high glucose conditions. Flux through the HBP under high glucose or glucosamine conditions also leads to an increase in insulin mRNA levels, probably by a mechanism involving increased MafA expression and MafA binding to the C1 element of the insulin promoter.