Central role for protein targeting to glycogen in the maintenance of cellular glycogen stores in 3T3-L1 adipocytes.

Overexpression of the protein phosphatase 1 (PP1) subunit protein targeting to glycogen (PTG) markedly enhances cellular glycogen levels. In order to disrupt the endogenous PTG-PP1 complex, small interfering RNA (siRNA) constructs against PTG were identified. Infection of 3T3-L1 adipocytes with PTG siRNA adenovirus decreased PTG mRNA and protein levels by >90%. In parallel, PTG reduction resulted in a >85% decrease in glycogen levels 4 days after infection, supporting a critical role for PTG in glycogen metabolism. Total PP1, glycogen synthase, and GLUT4 levels, as well as insulin-stimulated signaling cascades, were unaffected. However, PTG knockdown reduced glycogen-targeted PP1 protein levels, corresponding to decreased cellular glycogen synthase- and phosphorylase-directed PP1 activity. Interestingly, GLUT1 levels and acute insulin-stimulated glycogen synthesis rates were increased two- to threefold, and glycogen synthase activation in the presence of extracellular glucose was maintained. In contrast, glycogenolysis rates were markedly increased, suggesting that PTG primarily acts to suppress glycogen breakdown. Cumulatively, these data indicate that disruption of PTG expression resulted in the uncoupling of PP1 activity from glycogen metabolizing enzymes, the enhancement of glycogenolysis, and a dramatic decrease in cellular glycogen levels. Further, they suggest that reduction of glycogen stores induced cellular compensation by several mechanisms, but ultimately these changes could not overcome the loss of PTG expression.

Increased sensitivity of glycogen synthesis to phosphorylase-a and impaired expression of the glycogen-targeting protein R6 in hepatocytes from insulin-resistant Zucker fa/fa rats.

Hepatic insulin resistance in the leptin-receptor defective Zucker fa/fa rat is associated with impaired glycogen synthesis and increased activity of phosphorylase-a. We investigated the coupling between phosphorylase-a and glycogen synthesis in hepatocytes from fa/fa rats by modulating the concentration of phosphorylase-a. Treatment of hepatocytes from fa/fa rats and Fa/? controls with a selective phosphorylase inhibitor caused depletion of phosphorylase-a, activation of glycogen synthase and stimulation of glycogen synthesis. The flux-control coefficient of phosphorylase on glycogen synthesis was glucose dependent and at 10 mm glucose was higher in fa/fa than Fa/? hepatocytes. There was an inverse correlation between the activities of glycogen synthase and phosphorylase-a in both fa/fa and Fa/? hepatocytes. However, fa/fa hepatocytes had a higher activity of phosphorylase-a, for a corresponding activity of glycogen synthase. This defect was, in part, normalized by expression of the glycogen-targeting protein, PTG. Hepatocytes from fa/fa rats had normal expression of the glycogen-targeting proteins G(L) and PTG but markedly reduced expression of R6. Expression of R6 protein was increased in hepatocytes from Wistar rats after incubation with leptin and insulin. Diminished hepatic R6 expression in the leptin-receptor defective fa/fa rat may be a contributing factor to the elevated phosphorylase activity and/or its high control strength on glycogen synthesis.

Spatial reorganization of glycogen synthase upon activation in 3T3-L1 adipocytes.

The dephosphorylation of glycogen synthase is a key step in the stimulation of glycogen synthesis by insulin. To further investigate the hormonal regulation of glycogen synthase activity, enzymatic localization in 3T3-L1 adipocytes was determined by immunocytochemistry and confocal microscopy. In basal cells, glycogen synthase and the protein phosphatase-1-glycogen-targeting subunit, protein targeting to glycogen (PTG), were diffusely distributed throughout the cell. Insulin treatment had no effect on PTG distribution but resulted in a reorganization of glycogen synthase into punctate clusters. Glycogen synthase aggregation was restricted to discrete cellular sites, presumably where glycogen synthesis occurred. Omission of extracellular glucose or substitution with 2-deoxy-glucose blocked the insulin-induced redistribution of glycogen synthase. Addition of the glycogenolytic agent forskolin after insulin stimulation disrupted the clusters of glycogen synthase protein, restoring the immunostaining pattern to the basal state. Conversely, adenoviral-mediated overexpression of PTG resulted in the insulin-independent dephosphorylation of glycogen synthase and a redistribution of the enzyme from the cytosolic- to glycogen-containing fractions. The effects of PTG on glycogen synthase activity were mediated by multisite dephosphorylation, which was enhanced by insulin and 2-deoxy-glucose, and required a functional glycogen synthase-binding domain on PTG. However, PTG overexpression did not induce distinct glycogen synthase clustering in fixed cells, presumably because cellular glycogen levels were increased more than 7-fold under these conditions, resulting in a diffusion of sites where glycogen elongation occurred. Cumulatively, these data indicate that the hormonal regulation of glycogen synthesis rates in 3T3-L1 adipocytes is mediated in part through changes in the subcellular localization of glycogen synthase.

Epigallocatechin gallate induces expression of heme oxygenase-1 in endothelial cells via p38 MAPK and Nrf-2 that suppresses proinflammatory actions of TNF-α.

Epigallocatechin gallate (EGCG), the major polyphenol in green tea, acutely stimulates production of nitric oxide (NO) from vascular endothelium to reduce hypertension and improve endothelial dysfunction in spontaneously hypertensive rats. Herein, we explored additional mechanisms whereby EGCG may mediate beneficial cardiovascular actions. When compared with vehicle-treated controls, EGCG treatment (2.5 µM, 8 h) of human aortic endothelial cells (HAEC) caused a ~three-fold increase in heme oxygenase-1 (HO-1) mRNA and protein with comparable increases in HO-1 activity. This was unaffected by pretreatment of cells with wortmannin, LY294002, PD98059 or L-NAME (PI 3-kinase, MEK and NO synthase inhibitors, respectively). Pretreatment of HAEC with SB203580 (p38 MAPK inhibitor) or siRNA knockdown of p38 MAPK completely blocked EGCG-stimulated induction of HO-1. EGCG treatment also inhibited tumor-necrosis-factor-α-stimulated expression of vascular cell adhesion molecule (VCAM)-1 and decreased adhesion of monocytes to HAEC. siRNA knockdown of HO-1, p38 MAPK or Nrf-2 blocked these inhibitory actions of EGCG. In HAEC transiently transfected with a human HO-1 promoter luciferase reporter (or an isolated Nrf-2 responsive region), luciferase activity increased in response to EGCG. This was inhibitable by SB203580 pretreatment. EGCG-stimulated expression of HO-1 and Nrf-2 was blocked by siRNA knockdown of Nrf-2 or p38 MAPK. Finally, liver from mice chronically treated with EGCG had increased HO-1 and decreased VCAM-1 expression. Thus, in vascular endothelium, EGCG requires p38 MAPK to increase expression of Nrf-2 that drives expression of HO-1, resulting in increased HO-1 activity. Increased HO-1 expression may underlie anti-inflammatory actions of EGCG in vascular endothelium that may help mediate beneficial cardiovascular actions of green tea.

Generation of a dominant-negative glycogen targeting subunit for protein phosphatase-1.

Modulation of the expression of the protein phosphatase-1 (PP1) glycogen-targeting subunit PTG exerts profound effects on cellular glycogen metabolism in vitro and in vivo. PTG contains three distinct binding domains for glycogen, PP1, and a common site for glycogen synthase and phosphorylase. The impact of disrupting the PP1-binding domain on PTG function was examined in 3T3-L1 adipocytes. A full-length PTG mutant was generated as an adenoviral construct in which the valine and phenylalanine residues in the conserved PP1-binding domain were mutated to alanine (PTG-VF). Infection of fully differentiated 3T3-L1 adipocytes with the PTG-VF adenovirus reduced glycogen stores by over 50%. In vitro, PTG-VF competitively interfered with wild-type PTG action, suggesting that the mutant construct acted as a dominant-negative molecule. The reduction in cellular glycogen storage was due to a significantly increased rate of glycogen turnover. Interestingly, acute basal and insulin-stimulated glucose uptake and glycogen synthesis rates were enhanced in PTG-VF expressing cells vs. control 3T3-L1 adipocytes, likely as a compensatory response to the loss of glycogen stores. These results indicate that the mutation of the PP1-binding domain on PTG resulted in the generation of a dominant-negative molecule that impeded endogenous PTG action and reduced cellular glycogen levels, through enhancement of glycogenolysis rather than impairment of glycogen synthesis.

Krp1 (Sarcosin) promotes lateral fusion of myofibril assembly intermediates in cultured mouse cardiomyocytes.

Krp1, also called sarcosin, is a cardiac and skeletal muscle kelch repeat protein hypothesized to promote the assembly of myofibrils, the contractile organelles of striated muscles, through interaction with N-RAP and actin. To elucidate its role, endogenous Krp1 was studied in primary embryonic mouse cardiomyocytes. While immunofluorescence showed punctate Krp1 distribution throughout the cell, detergent extraction revealed a significant pool of Krp1 associated with cytoskeletal elements. Reduction of Krp1 expression with siRNA resulted in specific inhibition of myofibril accumulation with no effect on cell spreading. Immunostaining analysis and electron microscopy revealed that cardiomyocytes lacking Krp1 contained sarcomeric proteins with longitudinal periodicities similar to mature myofibrils, but fibrils remained thin and separated. These thin myofibrils were degraded by a scission mechanism distinct from the myofibril disassembly pathway observed during cell division in the developing heart. The data are consistent with a model in which Krp1 promotes lateral fusion of adjacent thin fibrils into mature, wide myofibrils and contribute insight into mechanisms of myofibrillogenesis and disassembly.

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

Adipocytes express the rate-limiting enzymes required for glycogen metabolism and increase glycogen synthesis in response to insulin. However, the physiological function of adipocytic glycogen in vivo is unclear, due in part to the low absolute levels and the apparent biophysical constraints of adipocyte morphology on glycogen accumulation. To further study the regulation of glycogen metabolism in adipose tissue, transgenic mice were generated that overexpressed the protein phosphatase-1 (PP1) glycogen-targeting subunit (PTG) driven by the adipocyte fatty acid binding protein (aP2) promoter. Exogenous PTG was detected in gonadal, perirenal, and brown fat depots, but it was not detected in any other tissue examined. PTG overexpression resulted in a modest redistribution of PP1 to glycogen particles, corresponding to a threefold increase in the glycogen synthase activity ratio. Glycogen synthase protein levels were also increased twofold, resulting in a combined greater than sixfold enhancement of basal glycogen synthase specific activity. Adipocytic glycogen levels were increased 200- to 400-fold in transgenic animals, and this increase was maintained to 1 yr of age. In contrast, lipid metabolism in transgenic adipose tissue was not significantly altered, as assessed by lipogenic rates, weight gain on normal or high-fat diets, or circulating free fatty acid levels after a fast. However, circulating and adipocytic leptin levels were doubled in transgenic animals, whereas adiponectin expression was unchanged. Cumulatively, these data indicate that murine adipocytes are capable of storing far higher levels of glycogen than previously reported. Furthermore, these results were obtained by overexpression of an endogenous adipocytic protein, suggesting that mechanisms may exist in vivo to maintain adipocytic glycogen storage at a physiological set point.

Glycogen branches out: new perspectives on the role of glycogen metabolism in the integration of metabolic pathways.

Glycogen is the storage form of carbohydrate for virtually every organism from yeast to primates. Most mammalian tissues store glucose as glycogen, with the major depots located in muscle and liver. The French physiologist Claude Bernard first identified a starch-like substance in liver and muscle and coined the term glycogen, or "sugar former," in the 1850s. During the 150 years since its identification, researchers in the field of glycogen metabolism have made numerous discoveries that are now recognized as significant milestones in biochemistry and cell signaling. Even so, more questions remain, and studies continue to demonstrate the complexity of the regulation of glycogen metabolism. Under classical definitions, the functions of glycogen seem clear: muscle glycogen is degraded to generate ATP during increased energy demand, whereas hepatic glycogen is broken down for release of glucose into the bloodstream to supply other tissues. However, recent findings demonstrate that the roles of glycogen metabolism in energy sensing, integration of metabolic pathways, and coordination of cellular responses to hormonal stimuli are far more complex.

Protein targeting to glycogen overexpression results in the specific enhancement of glycogen storage in 3T3-L1 adipocytes.

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 glycogen-metabolizing 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 microg/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 32P-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.

The glycogenic action of protein targeting to glycogen in hepatocytes involves multiple mechanisms including phosphorylase inactivation and glycogen synthase translocation.

Expression of the glycogen-targeting protein PTG promotes glycogen synthase activation and glycogen storage in various cell types. In this study, we tested the contribution of phosphorylase inactivation to the glycogenic action of PTG in hepatocytes by using a selective inhibitor of phosphorylase (CP-91149) that causes dephosphorylation of phosphorylase a and sequential activation of glycogen synthase. Similar to CP-91194, graded expression of PTG caused a concentration-dependent inactivation of phosphorylase and activation of glycogen synthase. The latter was partially counter-acted by the expression of muscle phosphorylase and was not additive with the activation by CP-91149, indicating that it is in part secondary to the inactivation of phosphorylase. PTG expression caused greater stimulation of glycogen synthesis and translocation of glycogen synthase than CP-91149, and the translocation of synthase could not be explained by accumulation of glycogen, supporting an additional role for glycogen synthase translocation in the glycogenic action of PTG. The effects of PTG expression on glycogen synthase and glycogen synthesis were additive with the effects of glucokinase expression, confirming the complementary roles of depletion of phosphorylase a (a negative modulator) and elevated glucose 6-phosphate (a positive modulator) in potentiating the activation of glycogen synthase. PTG expression mimicked the inactivation of phosphorylase caused by high glucose and counteracted the activation caused by glucagon. The latter suggests a possible additional role for PTG on phosphorylase kinase inactivation.

Expression of fatty acid binding proteins inhibits lipid accumulation and alters toxicity in L cell fibroblasts.

High levels of saturated, branched-chain fatty acids are deleterious to cells and animals, resulting in lipid accumulation and cytotoxicity. Although fatty acid binding proteins (FABPs) are thought to be protective, this hypothesis has not previously been examined. Phytanic acid (branched chain, 16-carbon backbone) induced lipid accumulation in L cell fibroblasts similar to that observed with palmitic acid (unbranched, C(16)): triacylglycerol >> free fatty acid > cholesterol > cholesteryl ester >> phospholipid. Although expression of sterol carrier protein (SCP)-2, SCP-x, or liver FABP (L-FABP) in transfected L cells reduced [(3)H]phytanic acid uptake (57-87%) and lipid accumulation (21-27%), nevertheless [(3)H]phytanic acid oxidation was inhibited (74-100%) and phytanic acid toxicity was enhanced in the order L-FABP >> SCP-x > SCP-2. These effects differed markedly from those of [(3)H]palmitic acid, whose uptake, oxidation, and induction of lipid accumulation were not reduced by L-FABP, SCP-2, or SCP-x expression. Furthermore, these proteins did not enhance the cytotoxicity of palmitic acid. In summary, intracellular FABPs reduce lipid accumulation induced by high levels of branched-chain but not straight-chain saturated fatty acids. These beneficial effects were offset by inhibition of branched-chain fatty acid oxidation that correlated with the enhanced toxicity of high levels of branched-chain fatty acid.