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...

The localized activation of circulating glucocorticoids in vivo by the enzyme 11␤-hydroxysteroid dehydrogenase type 1 (11␤-HSD1) plays a critical role in the development of the metabolic syndrome. However, the precise contribution of 11␤-HSD1 in the initiation of adipogenesis by inactive glucocorticoids is not fully understood. 3T3-L1 fibroblasts can be terminally differentiated to mature adipocytes in a glucocorticoid-dependent manner. Both inactive rodent dehydrocorticosterone and human cortisone were able to substitute for the synthetic glucocorticoid dexamethasone in 3T3-L1 adipogenesis, suggesting a potential role for 11␤-HSD1 in these effects. Differentiation of 3T3-L1 cells caused a strong increase in 11␤-HSD1 protein levels, which occurred late in the differentiation protocol. Reduction of 11␤-HSD1 activity in 3T3-L1 fibroblasts, achieved by pharmacological inhibition or adenovirally mediated delivery of short hairpin RNA constructs, specifically blocked the ability of inactive glucocorticoids to drive 3T3-L1 differentiation. However, even modest increases in exogenous 11␤-HSD1 expression in 3T3-L1 fibroblasts, to levels comparable with endogenous 11␤-HSD1 in differentiated 3T3-L1 adipocytes, were sufficient to block adipogenesis. Luciferase reporter assays indicated that overexpressed 11␤-HSD1 was catalyzing the inactivating dehydrogenase reaction, because the ability of both active and inactive glucocorticoids to activate the glucocorticoid receptor were largely suppressed. These results suggest that the temporal regulation of 11␤-HSD1 expression is tightly controlled in 3T3-L1 cells, so as to mediate the initiation of differentiation by inactive glucocorticoids and also to prevent the inhibitory activity of prematurely expressed 11␤-HSD1 during adipogenesis.