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.
Highlights
On the appropriate synthesis and secretion of the polypeptide hormone insulin, which is produced solely within the beta cells of the pancreas
The hexosamine biosynthetic pathway (HBP)2 is linked to glycolysis via fructose 6-phosphate, which is used by the gluta
We provide evidence that flux through the HBP and O-GlcNAcylation are involved in MafA expression induced by high glucose in beta cell lines
Summary
Chemicals—D-glucose, L-glucose, fructose, mannose, pyruvate, 2-deoxy-D-glucose (2DG), 6-deoxy-D-glucose, 3-O-methyl-D-glucose, glucosamine, insulin from bovine pancreas, potassium chloride (KCl), 6-diazo-5-oxo-L-norleucine (DON), and azaserine were obtained from Sigma. MIN6 cells were grown overnight (16–18 h) in Dulbecco’s modified Eagle’s medium containing D-glucose, L-glucose, fructose, mannose, or pyruvate as indicated in the figure legends. The data are expressed as -fold increases in mRNA levels in cells cultured under the conditions described in each figure legend (such as 25 mM glucose) over the amount of mRNA levels in cells treated with pyruvate or 1 mM glucose. The amount of insulin secreted (ng/ ml), normalized to total protein levels (mg/ml), was calculated, and the data were expressed as -fold changes over the amount of insulin secreted (ng/ml insulin versus mg/ml total protein) from MIN6 cells incubated with 1 mM glucose. The released intracellular ATP was measured using the ATP bioluminescent somatic cell assay kit (Sigma) according to the manufacturer’s instructions and as described for INS-1 beta cells [45]. TBP was used as a loading control for Western blots
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
