Glycogen biogenesis in rat 1 fibroblasts expressing rabbit muscle glycogenin.

Abstract

Glycogenin, a self-glucosylating protein involved in the initiation of glycogen biosynthesis, varies in intracellular concentration from barely detectable in liver to a high level in muscle. The effect of increasing the glycogenin level on glycogen synthesis was studied in rat 1 fibroblasts stably overexpressing rabbit muscle glycogenin. In the presence of glucose, all of the expressed glycogenin was attached to polysaccharide and the free protein could only be detected by western blot analysis after incubation of cells in a glucose-depleted medium or treatment of the cell extract with alpha-amylase. In control cells, increased extracellular glucose concentrations promoted translocation of glycogen synthase from the soluble to the pellet fraction with an increase in the associated glycogen. Overexpression of glycogenin did not affect total intracellular glycogen and glycogen synthase levels at any concentration of glucose but significantly reduced glucose-induced accumulation of insoluble glycogen and translocation of glycogen synthase. Immunofluorescence analysis revealed a diffuse cytoplasmic distribution of glycogenin expressed in rat 1 cells. In rat 1 cells incubated with glucose, discrete deposits of glycogen were detected by staining with HIO4/Schiff but this was eliminated by overexpressing glycogenin. Analysis of [14C]glucose- or [35S]methionine-labeled extracts from glycogenin-expressing cells by continuous polyacrylamide gel electrophoresis and by two-dimensional gel electrophoresis revealed a continuum of glycogenin-containing species from low molecular mass to sizes significantly greater than 400 kDa. We conclude that (a) overexpression of glycogenin does not enhance glycogen synthesis but causes production of more, smaller, glycogen molecules with a concomitant change in their intracellular localization; (b) glycogenin and elevated glucose have opposing effects on the distribution of glycogenin and glycogen synthase in rat 1 cells; and (c) the biogenesis of glycogen in rat 1 cells occurs without the accumulation of any major intermediate form.