Intracellular distribution of glycogen synthase and glycogen in primary cultured rat hepatocytes

Abstract

Changes in the intracellular distribution of liver glycogen synthase (GS) might constitute a new regulatory mechanism for the activity of this enzyme at cellular level. Our previous studies indicated that incubation of isolated hepatocytes with glucose activated GS and resulted in its translocation from a homogeneous cytosolic distribution to the cell periphery. These studies also suggested a relationship with insoluble elements of the cytoskeleton, in particular actin. Here we show the translocation of GS in a different experimental model that allows the analysis of this phenomenon in long-term studies. We describe the reversibility of translocation of GS and its effect on glycogen distribution. Incubation of cultured rat hepatocytes with glucose activated GS and triggered its translocation to the hepatocyte periphery. The relative amount of the enzyme concentrated near the plasma membrane increased with time up to 8 h of incubation with glucose, when the glycogen stores reached their maximal value. The lithium-induced covalent activation of GS was not sufficient to cause its translocation to the cell periphery. The intracellular distribution of GS closely resembled that of glycogen. Our results showed an interaction between GS and an insoluble element of the hepatocyte matrix. Although no co-localization between actin filaments and GS was observed in any condition, disruption of actin cytoskeleton resulted in a significantly lower percentage of cells in which the enzyme translocated to the cell periphery in response to glucose. This observation suggests that the microfilament network has a role in the translocation of GS.

Glucose 6-Phosphate Produced by Gluconeogenesis and by Glucokinase Is Equally Effective in Activating Hepatic Glycogen Synthase

“…Immunofluorescence analysis, using an antibody that specifically recognizes liver GS (6), showed that incubation with DHA produced an accumulation of the enzyme at the periphery of the hepatocytes, similar to that observed after glucose treatment. In control cells, GS was distributed throughout the cytoplasm, and, after incubation with glucose or DHA, the enzyme formed patches near the hepatocyte plasma membrane ( Fig.…”

“…First, the mere activation of GS induced by treatment with lithium chloride, a known inhibitor of glycogen synthase kinase-3 (42,43), is not sufficient to cause GS translocation to the hepatocyte periphery (6). Second, the common metabolites in the biosynthetic pathway of glycogen from glucose and from gluconeogenic precursors are Glc-6-P, which is in equilibrium with Glc-1-P, and UDP-Glc, the substrate of GS and immediate precursor in glycogen synthesis.…”

“…Cells were incubated for 16 h with DMEM without glucose, treated for 2 h with 25 mM glucose, and finally collected for measurement of GK and HK, G6Pase and total GS activities as described under "Experimental Procedures." Activities are expressed in milliunits/10 6 …”

Liver Glycogen Synthase but Not the Muscle Isoform Differentiates between Glucose 6-Phosphate Produced by Glucokinase or Hexokinase

“…MGS is also concentrated in the nucleus at low glucose and translocates to the cytosol, where it adopts a particulate pattern, at high glucose concentrations (5,14). In contrast, LGS presents a cytosolic distribution in the absence of glucose and concentrates at the periphery of the hepatocyte when the concentration of the hexose increases (27). Finally HK I has been shown to reversibly bind to the outer mitochondrial membrane through a hydrophobic Nterminal sequence (28).…”

Genetic models rule out a major role of beta cell glycogen in the control of glucose homeostasis

“…Protein was quantified using the Bradford assay (Bio-Rad, Hercules, CA, USA). Proteins were separated in a 10% SDS-PAGE gel, transferred to a PVDF membrane, and immunoblotted with rabbit anti-GS 3893 (1:1,000, Cell Signaling, Danvers, MA, USA), rabbit anti-muscle GS MGS3 [23], rabbit anti-liver GS L1 [24], mouse anti-actin AC-40 (1:25,000, Abcam, Cambridge, UK) and horseradish peroxidase-linked (anti-rabbit from GE, Little Chalfont, UK and anti-mouse from Dako, Glostrup, Denmark) secondary antibodies. Antibodies were validated using tissue/cell protein extracts where the antigen in question is not expressed.…”