Glucose Metabolism to Glucosamine Is Necessary for Glucose Stimulation of Transforming Growth Factor-α Gene Transcription

Peter P Sayeski,Jeffrey E Kudlow

doi:10.1074/jbc.271.25.15237

Abstract

Transforming growth factor-alpha (TGFalpha) gene transcription can be increased when arterial smooth muscle cells are exposed to supraphysiological concentrations of glucose, and this effect of glucose can be mimicked by glucosamine. To determine whether the metabolism of glucose to glucosamine is required for this glucose effect, the rate-limiting step in glucose metabolism to glucosamine through the enzyme glutamine:fructose-6-phosphate amidotransferase (GFAT) was blocked using pharmacological and antisense strategies. We found that blockage of GFAT activity or expression significantly blunted the glucose-induced increase of TGFalpha expression. Blockage of GFAT also resulted in a decreased RL2 signal on intracellular proteins as detected by Western blotting and indirect immunofluorescence. The RL2 monoclonal antibody recognizes an epitope on proteins that contain N-acetylglucosamine and thus is a measure of protein glycosylation. Conversely, treatment of the cells with glucose and glucosamine resulted in an increase in the RL2 epitope on intracellular proteins. These results indicate that the metabolism of glucose to glucosamine is necessary for the transcriptional stimulation of TGFalpha expression in vascular smooth muscle cells by glucose. Furthermore, the level of glycosylation of some intracellular proteins can be modulated in response to physiological changes in the extracellular glucose concentration and the net activity of GFAT.

Highlights

Many of the long term complications of diabetes mellitus relate to vascular pathology, and in type I diabetes, there is strong epidemiologic evidence linking hyperglycemia to vascular disease [1, 2]
This property of RL2 allowed us to obtain a measure of the GlcNAc content of proteins in individual cells by immunofluorescence microscopy providing an assessment of the Glucose Induction of TGF␣ Transcription downstream consequences of changes in glucosamine synthesis resulting from blockade of glutamine:fructose-6-phosphate amidotransferase (GFAT) activity or expression
We can conclude that glucosamine synthesis from glucose is necessary for the induction of TGF␣ gene transcription by glucose and that changes in the ambient concentration of glucose through physiologically relevant concentrations does result in changes in the glycosylation of intracellular and nuclear proteins

Summary

Introduction

Many of the long term complications of diabetes mellitus relate to vascular pathology, and in type I diabetes, there is strong epidemiologic evidence linking hyperglycemia to vascular disease [1, 2]. The addition of glucosamine to the cell culture medium [10] and overexpression of GFAT [9] both result in an increase in the intracellular concentration of uridine diphosphate-Nacetylglucosamine (UDP-GlcNAc), a substrate for protein glycosylation.

Results

Conclusion