Protein Targeting to Glycogen Overexpression Results in the Specific Enhancement of Glycogen Storage in 3T3-L1 Adipocytes

Abstract

Protein phosphatase-1 (PP1) plays an important role in the regulation of glycogen synthesis by insulin. Protein targeting to glycogen (PTG) enhances glycogen accumulation by increasing PP1 activity against glycogenmetabolizing enzymes. However, the specificity of PTG's effects on cellular dephosphorylation and glucose metabolism is unclear. Overexpression of PTG in 3T3-L1 adipocytes using a doxycycline-controllable adenoviral construct resulted in a 10 -20-fold increase in PTG levels and an 8-fold increase in glycogen levels. Inclusion of 1 g/ml doxycycline in the media suppressed PTG expression, and fully reversed all PTG-dependent effects. Infection of 3T3-L1 adipocytes with the PTG adenovirus caused a marked dephosphorylation and activation of glycogen synthase. The effects of PTG seemed specific, because basal and insulin-stimulated phosphorylation of a variety of signaling proteins was unaffected. Indeed, glycogen synthase was the predominant protein whose phosphorylation state was decreased in 32 P-labeled cells. PTG overexpression did not alter PP1 protein levels but increased PP1 activity 6-fold against phosphorylase in vitro. In contrast, there was no change in PP1 activity measured using myelin basic protein, suggesting that PTG overexpression specifically directed PP1 activity against glycogen-metabolizing enzymes. To investigate the metabolic consequences of altering PTG levels, glucose uptake and storage in 3T3-L1 adipocytes was measured. PTG overexpression did not affect 2-deoxy-glucose transport rates in basal and insulin-stimulated cells but dramatically enhanced glycogen synthesis rates under both conditions. Despite the large increases in cellular glucose flux upon PTG overexpression, basal and insulin-stimulated glucose incorporation into lipid were unchanged. Cumulatively, these data indicate that PTG overexpression in 3T3-L1 adipocytes discretely stimulates PP1 activity against glycogen synthase and phosphorylase, resulting in a marked and specific increase in glucose uptake and storage as glycogen.Insulin exerts its anabolic actions by promoting the uptake and storage of glucose and lipids in target tissues. Insulin coordinates the stimulation of the translocation of both glucose and fatty acid transporters to the cell surface (1, 2) and the modulation of the activity of key metabolic enzymes (3). In addition, insulin inhibits hepatic glycogenolysis and adipocytic triglyceride breakdown, further lowering excess glucose and free fatty acids levels in the bloodstream. Disruption of the complex interplay between lipid and carbohydrate metabolism results in the development of insulin resistance and type II diabetes (4, 5). However, the molecular mechanisms by which insulin potently regulates energy uptake and storage in vivo are not fully understood.Glycogen synthase activity is stimulated by insulin in liver, muscle, and adipose tissue via protein dephosphorylation, allosteric activation, and enzymatic translocation. Glycogen synthase is phosphorylated on up to nine residues by a variety of ki...

Transgenic overexpression of protein targeting to glycogen markedly increases adipocytic glycogen storage in mice

“…One such protein, termed protein targeting to glycogen (PTG), was first identified from 3T3-L1 adipocytes (57) and remains the only PP1 glycogen-targeting subunit reported to be expressed in adipocytes. Importantly, as reported by several groups, overexpression of PTG in a variety of cell types in vitro and in rodent liver in vivo markedly enhanced cellular glycogen levels (17,20,22,24,37,50,57,72).…”

The Glycogenic Action of Protein Targeting to Glycogen in Hepatocytes Involves Multiple Mechanisms Including Phosphorylase Inactivation and Glycogen Synthase Translocation

“…Hepatocyte monolayers were incubated with the PTG-adenovirus (75-1800 pfu/ cell). Where indicated, cells were cultured in medium supplemented with 1 g/ml doxycycline to inhibit PTG expression (21). To confirm transfection efficiency, cells were treated with an adenovirus encoding ␤-galactosidase and stained (22).…”

“…Preparation of Recombinant Adenoviruses-PTG was subcloned into the tetracycline-responsive Adeno-X DNA vector (21). The PTG-adenovirus and the regulatory adenovirus (Adeno-X Tet-Off) were diluted in MEM containing 0.5 g/ml poly-L-lysine hydrobromide.…”

“…The effects of glucose and PTG were not additive suggesting that PTG mimics the effect of glucose. DISCUSSION PTG expression activates glycogen synthase and stimulates glycogen storage in various cell types (8,12,13,21). Because phosphorylase a has major roles in the control of synthase phosphatase (1) and the rate of glycogen synthesis in hepatocytes (15, 29), we tested the role of inactivation of phosphorylase in the glycogenic action of PTG in hepatocytes.…”

Expression of a synapsin IIb site 1 phosphorylation mutant in 3T3-L1 adipocytes inhibits basal intracellular retention of Glut4

“…Adenovirus was purified from packaging cell lysates using Optiprep gradients following the manufacturer's recommended protocol for retroviral purification (Axis-Shield UK, Dundee, UK). Adipocytes and fibroblasts were infected with adenovirus as previously described with modifications as noted (Greenberg et al, 2003;Sanguinetti et al, 2003). Adipocytes were infected on day 7 of differentiation (1 day after removal of insulin).…”