Glycogen-targeting Subunit Of Protein Phosphatase-1 Research Articles

Ablation of PPP1R3G reduces glycogen deposition and mitigates high-fat diet induced obesity

Glycogen and triglyceride are two major forms of energy storage in the body and provide the fuel during different phases of food deprivation. However, how glycogen metabolism is linked to fat deposition in adipose tissue has not been clearly characterized. We generated a mouse model with whole-body deletion of PPP1R3G, a glycogen-targeting subunit of protein phosphatase-1 required for glycogen synthesis. Upon feeding with high-fat diet, the body weight and fat composition are significantly reduced in the PPP1R3G−/- mice compared to the wild type controls. The metabolic rate of the mice as measured by O2 consumption and CO2 production is accelerated by PPP1R3G deletion. The high-fat diet-induced liver steatosis is also slightly relieved by PPP1R3G deletion. The glycogen level in adipose tissue is reduced by PPP1R3G deletion. In 3T3L1 cells, overexpression of PPP1R3G leads to increases of both glycogen and triglyceride levels. In conclusion, our study indicates that glycogen is actively involved in fat accumulation in adipose tissue and obesity development upon high-fat diet. Our study also suggests that PPP1R3G is an important player that links glycogen metabolism to lipid metabolism in vivo.

Molecular Recognition of the Protein Phosphatase 1 Glycogen Targeting Subunit by Glycogen Phosphorylase

Disrupting the interaction between glycogen phosphorylase and the glycogen targeting subunit (G(L)) of protein phosphatase 1 is emerging as a novel target for the treatment of type 2 diabetes. To elucidate the molecular basis of binding, we have determined the crystal structure of liver phosphorylase bound to a G(L)-derived peptide. The structure reveals the C terminus of G(L) binding in a hydrophobically collapsed conformation to the allosteric regulator-binding site at the phosphorylase dimer interface. G(L) mimics interactions that are otherwise employed by the activator AMP. Functional studies show that G(L) binds tighter than AMP and confirm that the C-terminal Tyr-Tyr motif is the major determinant for G(L) binding potency. Our study validates the G(L)-phosphorylase interface as a novel target for small molecule interaction.

A novel glycogen-targeting subunit of protein phosphatase 1 that is regulated by insulin and shows differential tissue distribution in humans and rodents.

Stimulation of glycogen-targeted protein phosphatase 1 (PP1) activity by insulin contributes to the dephosphorylation and activation of hepatic glycogen synthase (GS) leading to an increase in glycogen synthesis. The glycogen-targeting subunits of PP1, GL and R5/PTG, are downregulated in the livers of diabetic rodents and restored by insulin treatment. We show here that the mammalian gene PPP1R3E encodes a novel glycogen-targeting subunit of PP1 that is expressed in rodent liver. The phosphatase activity associated with R3E is slightly higher than that associated with R5/PTG and it is downregulated in streptozotocin-induced diabetes by 60-70% and restored by insulin treatment. Surprisingly, although mRNA for R3E is most highly expressed in rat liver and heart muscle, with only low levels in skeletal muscle, R3E mRNA is most abundant in human skeletal muscle and heart tissues with barely detectable levels in human liver. This species-specific difference in R3E mRNA expression has similarities to the high level of expression of GL mRNA in human but not rodent skeletal muscle. The observations imply that the mechanisms by which insulin regulates glycogen synthesis in liver and skeletal muscle are different in rodents and humans.

Role of the liver in the control of glucose-lipid utilization and body weight.

This review depicts recent developments concerning the role of the liver in control of carbohydrate and lipid utilization from ingestion to storage; it covers the liver's influence on food intake, post-absorptive nutrient metabolism and body weight. The mechanisms involved have implications for the pathogenesis of obesity and type II diabetes. Recent studies have identified some of the molecular and biochemical mechanisms which control whole body and hepatic carbohydrate and lipid metabolism, thus providing the basis of the liver's role in the control of food intake, metabolism, and body weight. Fatty acids are known to effect gene transcription in various ways. Advances in our understanding of the control of glucose and lipid utilization by the liver include (1). a better functional characterization of some newly discovered transcription factors, (2). new discoveries concerning the physiological and pathophysiological role of hepatic glucokinase and of the glycogen-targeting subunits of protein phosphatase-1, and (3). the demonstration of substantial overlap in the molecular control mechanisms of glucose-lipid utilization. Also, impaired insulin signaling due to a certain gene (Foxo1) has emerged as a possible unifying mechanism for various common metabolic abnormalities of type II diabetes. Finally, recent findings confirm and extend previous knowledge about the important role of hepatic nerves in the control of liver and whole body glucose-lipid utilization. The identification of new molecular and neural mechanisms of the hepatic control of glucose-lipid utilization and body weight provides a focus for future studies and may eventually help to develop new treatments for obesity and type II diabetes.

Hepatic Expression of a Targeting Subunit of Protein Phosphatase-1 in Streptozotocin-diabetic Rats Reverses Hyperglycemia and Hyperphagia Despite Depressed Glucokinase Expression

Glycogen-targeting subunits of protein phosphatase-1 (PP-1) are scaffolding proteins that facilitate the regulation of key enzymes of glycogen metabolism by PP-1. In the current study, we have tested the effects of hepatic expression of GMDeltaC, a truncated version of the muscle-targeting subunit isoform, in rats rendered insulin-deficient via injection of a single moderate dose of streptozotocin (STZ). Three key findings emerged. First, GMDeltaC expression in liver was sufficient to fully normalize blood glucose levels (from 335 +/- 31 mg/dl prior to viral injection to 109 +/- 28 mg/dl 6 days after injection) and liver glycogen content in STZ-injected rats. Second, this normalization occurred despite very low levels of liver glucokinase expression in the insulin-deficient STZ-injected rats. Finally, the hyperphagia induced by STZ injection was completely reversed by GMDeltaC expression in liver. In contrast to these findings with GMDeltaC, overexpression of another targeting subunit, GL, in STZ-injected rats caused a large increase in liver glycogen stores but only a transient decrease in food intake and blood glucose levels. The surprising demonstration of a glucose-lowering effect of GMDeltaC in the background of depressed hepatic glucokinase expression suggests that controlled stimulation of liver glycogen storage may be an effective mechanism for improving glucose homeostasis, even when normal pathways of glucose disposal are impaired.

Reversal of Diet-induced Glucose Intolerance by Hepatic Expression of a Variant Glycogen-targeting Subunit of Protein Phosphatase-1

Glycogen-targeting subunits of protein phosphatase-1 facilitate interaction of the phosphatase with enzymes of glycogen metabolism. Expression of one family member, PTG, in the liver of normal rats improves glucose tolerance without affecting other plasma variables but leaves animals unable to reduce hepatic glycogen stores in response to fasting. In the current study, we have tested whether expression of other targeting subunit isoforms, such as the liver isoform G(L), the muscle isoform G(M)/R(Gl), or a truncated version of G(M)/R(Gl) termed G(M)DeltaC in liver ameliorates glucose intolerance in rats fed on a high fat diet (HF). HF animals overexpressing G(M)DeltaC, but not G(L) or G(M)/R(Gl), exhibited a decline in blood glucose of 35-44 mg/dl relative to control HF animals during an oral glucose tolerance test (OGTT) such that levels were indistinguishable from those of normal rats fed on standard chow at all but one time point. Hepatic glycogen levels were 2.1-2.4-fold greater in G(L)- and G(M)DeltaC-overexpressing HF rats compared with control HF animals following OGTT. In a second set of studies on fed and 20-h fasted HF animals, G(M)DeltaC-overexpressing rats lowered their liver glycogen levels by 57% (from 402 +/- 54 to 173 +/- 27 microg of glycogen/mg of protein) in the fasted versus fed states compared with only 44% in G(L)-overexpressing animals (from 740 +/- 35 to 413 +/- 141 microg of glycogen/mg of protein). Since the OGTT studies were performed on 20-h fasted rats, this meant that G(M)DeltaC-overexpressing rats synthesized much more glycogen than G(L)-overexpressing HF rats during the OGTT (419 versus 117 microg of glycogen/mg of protein, respectively), helping to explain why G(M)DeltaC preferentially enhanced glucose clearance. We conclude that G(M)DeltaC has a unique combination of glycogenic potency and responsiveness to glycogenolytic signals that allows it to be used to lower blood glucose levels in diabetes.

Glycogen-targeting Subunits and Glucokinase Differentially Affect Pathways of Glycogen Metabolism and Their Regulation in Hepatocytes

Overexpression of the glucose-phosphorylating enzyme glucokinase (GK) or members of the family of glycogen-targeting subunits of protein phosphatase-1 increases hepatic glucose disposal and glycogen synthesis. This study was undertaken to evaluate the functional properties of a novel, truncated glycogen-targeting subunit derived from the skeletal muscle isoform G(M)/R(Gl) and to compare pathways of glycogen metabolism and their regulation in cells with overexpressed targeting subunits and GK. When overexpressed in hepatocytes, truncated G(M)/R(Gl) (G(M)DeltaC) was approximately twice as potent as full-length G(M)/R(Gl) in stimulation of glycogen synthesis, but clearly less potent than GK or two other native glycogen-targeting subunits, G(L) and PTG. We also found that cells with overexpressed G(M)DeltaC are unique in that glycogen was efficiently degraded in response to lowering of media glucose concentrations, stimulation with forskolin, or a combination of both maneuvers, whereas cells with overexpressed G(L), PTG, or GK exhibited impairment in one or both of these glycogenolytic signaling pathways. (2)H NMR analysis of purified glycogen revealed that hepatocytes with overexpressed GK synthesized a larger portion of their glycogen from triose phosphates and a smaller portion from tricarboxylic acid cycle intermediates than cells with overexpressed glycogen-targeting subunits. Additional evidence for activation of distinct pathways of glycogen synthesis by GK and targeting subunits is provided by the additive effect of co-overexpression of the two types of proteins upon glycogen synthesis and a much larger stimulation of glucose utilization, glucose transport, and lactate production elicited by GK. We conclude that overexpression of the novel targeting subunit G(M)DeltaC confers unique regulation of glycogen metabolism. Furthermore, targeting subunits and GK stimulate glycogen synthesis by distinct pathways.

Overexpression of the Glycogen Targeting (GM) Subunit of Protein Phosphatase-1

The GM glycogen-targeting subunit of protein phosphatase-1 (PP1) is believed to be involved in dephosphorylation of the enzymes of glycogen metabolism. To assess the roles of GM on glycogen metabolism, we created site-directed GM mutants and overexpressed them in Chinese hamster ovary (CHO) cells expressing human insulin receptor. Overexpressed GM recruited glycogen synthase as well as PP1 to the glycogen pellet, and upregulated basal glycogen synthase activity. Overexpressed GM-67A (Ser-67 replaced with alanine) exhibited decreased sensitivity to suppression of glycogen synthase activity by forskolin, while overexpression of GM-48A (Ser-48 replaced with alanine) preserved glycogen synthase activation in response to insulin. These observations indicate that in CHO cells overexpressing GM; (1) GM translocates glycogen synthase to the glycogen pellet and affected basal glycogen synthase, (2) Ser-67 might be involved in the suppression of glycogen synthase activity by glycogenolytic agents, and (3) Ser-48 might not commit to activation of glycogen synthase by insulin.

Distinctive Regulatory and Metabolic Properties of Glycogen-targeting Subunits of Protein Phosphatase-1 (PTG, GL, GM/RGl) Expressed in Hepatocytes

Glycogen-targeting subunits of protein phosphatase-1 facilitate interaction of the phosphatase with enzymes of glycogen metabolism. We have shown that overexpression of one member of the family, protein targeting to glycogen (PTG), causes large increases in glycogen storage in isolated hepatocytes or intact rat liver. In the current study, we have compared the metabolic and regulatory properties of PTG (expressed in many tissues), with two other members of the gene family, G(L) (expressed primarily in liver) and G(M)/R(Gl) (expressed primarily in striated muscle). Adenovirus-mediated expression of these proteins in hepatocytes led to the following key observations. 1) G(L) has the highest glycogenic potency among the three forms studied. 2) Glycogen synthase activity ratio is much higher in G(L)-overexpressing cells than in PTG or G(M)/R(Gl)-overexpressing cells. Thus, at moderate levels of G(L) overexpression, glycogen synthase activity is increased by insulin treatment, but at higher levels of G(L) expression, insulin is no longer required to achieve maximal synthase activity. In contrast, cells with high levels of PTG overexpression retain dose-dependent regulation of glycogen synthesis and glycogen synthase enzyme activity by insulin. 3) G(L)- and G(M)/R(Gl)-overexpressing cells exhibit a strong glycogenolytic response to forskolin, whereas PTG-overexpressing cells are less responsive. This difference may be explained in part by a lesser forskolin-induced increase in glycogen phosphorylase activity in PTG-overexpressing cells. Based on these results, we suggest that expression of either G(L) or G(M)/R(Gl) in liver of diabetic animals may represent a strategy for lowering of blood glucose levels in diabetes.

Activation of direct and indirect pathways of glycogen synthesis by hepatic overexpression of protein targeting to glycogen.

Glycogen-targeting subunits of protein phosphatase-1, such as protein targeting to glycogen (PTG), direct the phosphatase to the glycogen particle, where it stimulates glycogenesis. We have investigated the metabolic impact of overexpressing PTG in liver of normal rats. After administration of PTG cDNA in a recombinant adenovirus, animals were fasted or allowed to continue feeding for 24 hours. Liver glycogen was nearly completely depleted in fasted control animals, whereas glycogen levels in fasted or fed PTG-overexpressing animals were 70% higher than in fed controls. Nevertheless, transgenic animals regulated plasma glucose, triglycerides, FFAs, ketones, and insulin normally in the fasted and fed states. Fasted PTG-overexpressing animals receiving an oral bolus of [U-(13)C]glucose exhibited a large increase in hepatic glycogen content and a 70% increase in incorporation of [(13)C]glucose into glycogen. However, incorporation of labeled glucose accounted for only a small portion of the glycogen synthesized in PTG-overexpressing animals, consistent with our earlier finding that PTG promotes glycogen synthesis from gluconeogenic precursors. We conclude that hepatic PTG overexpression activates both direct and indirect pathways of glycogen synthesis. Because of its ability to enhance glucose storage without affecting other metabolic indicators, the glycogen-targeting subunit may prove valuable in controlling blood glucose levels in diabetes.

Role of Protein Targeting to Glycogen (PTG) in the Regulation of Protein Phosphatase-1 Activity

We have recently cloned from 3T3-L1 adipocytes a novel glycogen-targeting subunit of protein phosphatase-1, termed PTG (Printen, J. A., Brady, M. J., and Saltiel, A. R. (1997) Science 275, 1475-1478). Differentiation of 3T3-L1 fibroblasts into highly insulin-responsive adipocytes resulted in a marked increase in PTG expression. Immobilized glutathione S-transferase (GST)-PTG fusion protein specifically bound either PP1 or phosphorylase a. Addition of soluble GST-PTG to 3T3-L1 lysates increased PP1 activity against 32P-labeled phosphorylase a by decreasing the Km of PP1 for phosphorylase 5-fold, while having no effect on the Vmax of the dephosphorylation reaction. Alternatively, PTG did not affect PP1 activity against hormone-sensitive lipase. PTG was not a direct target of intracellular signaling, as insulin or forskolin treatment of cells did not activate a kinase capable of phosphorylating PTG in vivo or in vitro. Finally, PTG decreased the ability of DARPP-32 to inhibit PP1 activity from 3T3-L1 adipocyte lysates. These data cumulatively suggest that PTG increases PP1 activity against specific proteins by several distinct mechanisms.