Matthew Brady, Ph.D.

Role of Protein Phosphatase-1 and Glycogen Targeting Subunits in Insulin Metabolic Signaling

Research Summary

Hormonal Regulation of Glycogen Synthesis

Insulin is the most-potent physiological anabolic agent known, promoting the synthesis and storage of carbohydrates and lipids, and inhibiting their degradation and release into the circulation. This action of the hormone is due in part to the acute regulation of metabolic enzymes through changes in their phosphorylation state. In fat, liver, and muscle, insulin stimulates the dephosphorylation of a number of enzymes involved in glycogen and lipid metabolism via activation of protein phosphatase-1 (PP1). Although PP1 is a cytosolic protein, the phosphatase is compartmentalized throughout the cell by discrete targeting subunits. These proteins confer substrate specificity to PP1 and mediate the specific regulation of intracellular pools of PP1 by a variety of extracellular signals. The main focus of the laboratory is the study of the hormonal regulation of glycogen metabolism. We have identified a novel PP1 regulatory subunit, termed PTG for Protein Targeting to Glycogen. This molecule binds to PP1 and glycogen, thus targeting the phosphatase to the glycogen particle. Additionally, PTG specifically binds to several PP1 substrates that are key enzymatic regulators of glycogen metabolism. Overexpression of PTG in cultured cells and intact animals causes the intracellular redistribution of PP1 and glycogen metabolizing enzymes, and a marked increase in glycogen stores. These results suggest that PTG acts as a molecular scaffold, assembling PP1 with specific substrate proteins, allowing for the efficient hormonal regulation of glycogen metabolism.

We are interested in the effects of modulating glycogen levels on insulin secretion from pancreatic b-cells and insulin metabolic signaling. Initial efforts are directed at adenoviral-mediated overexpression of wild type, super active and potential dominant negative PTG constructs in insulin secreting cell lines. The principal question is whether modulating the glycogen synthetic capacity of these cells will affect glucose-induced insulin secretion. Can glucose be shunted from glycolysis and ATP production to glycogen storage? If so, will PTG overexpression cause a right shift in the glucose dose response curve for insulin secretion? Conversely, what are the effects of disrupting glycogen metabolism on Òglucose sensingÓ by the beta cell? Future plans include generation of transgenic mouse lines with beta cell specific expression of PTG constructs, and examination of islet differentiation and function in vivo.

A second major project in the lab is the examination of the interdependency of intracellular glycogen levels and insulin metabolic signaling in 3T3-L1 adipocytes. By using adenoviral vectors, several PTG constructs will be over expressed in these cells. The effects of elevation or depletion of cellular glycogen on insulin-regulated glucose uptake and storage will then be examined. Since lipid and glycogen are the principal forms of carbon storage in mammals, a high priority is the adipose specific expression of PTG constructs in transgenic animals. Can adipocytes be made to shunt more stored energy from lipid to glycogen? What is the physiological role of glycogen metabolism in fat cells, especially with regards to secretion of a variety of factors such as leptin, resistin, TNFa and Acrp30? And most importantly, what are the ramifications of altering adipose tissue glycogen stores (up or down) on whole animal metabolism, diet induced obesity and insulin resistance?

Finally, the molecular mechanisms by which insulin regulates glycogen metabolism remain poorly understood. In addition to PTG, three other proteins have been described that target PP1 to the glycogen particle. Despite a proposed common function, this family of four proteins is not highly conserved and displays an overlapping tissue distribution. Overexpression studies in cell lines or animals reveal major differences among these proteins regarding basal glycogen levels and hormonal responsiveness. To better understand the unique properties each protein may confer on glycogen-targeted PP1 activity, a series of chimeric constructs has been generated. The putative regulatory sites of two of these proteins have been introduced into PTG, and conversely deleted from the native molecules. Future plans involve overexpression of the wild type and chimeric targeting subunit molecules in primary hepatocytes and skeletal muscle cells. The hormonal regulation of glycogen synthesis and degradation will be studied, in an effort to better understand the unique roles that each of these PP1 targeting subunits play in glycogen metabolism.

Together, these studies will generate new insight into the hormonal regulation of glycogen synthesis and the potential role for altered glycogen metabolism in the development and progression of type I and II diabetes.

Selected Papers

Printen JA, Brady MJ and Saltiel AR (1997). PTG, a protein phosphatase 1-binding protein with a role in glycogen metabolism. Science 275, 1475-1478

Newgard CB, Brady MJ, O'Doherty RM and Saltiel AR (2000). Organizing glucose disposal: the emerging roles of the glycogen targeting subunits of protein phosphatase-1. Diabetes 49, 1967-1977

Fong NM, Jensen TC, Shah AM, Parekh NN, Saltiel AR and Brady MJ (2000). Identification of binding sites on PTG for enzymes of glycogen metabolism. J. Biol. Chem. 275, 35034-35039

Jensen TC, Crosson SM, Kartha PM and Brady MJ (2000). Specific desensitization of glycogen synthase activation by insulin in 3T3-L1 adipocytes: connection between enzymatic activation and subcellular localization. J. Biol. Chem. 275, 40148-40154

Brady MJ and Saltiel AR (2001). The role of protein phosphatase-1 in insulin action. Recent Progress Horm. Res. 56, 157-173

Yan L, Nairn AC, Palfrey HC and Brady MJ (2003). Glucose regulates EF-2 phosphorylation and protein translation by a protein phosphatase-2A-dependent mechanism in INS-1-derived 832/13 cells. J. Biol. Chem. 278, 18177-18183

Greenberg CC, Meredith KN, Yan L and Brady MJ (2003). Protein targeting to glycogen overexpression results in the specific enhancement of glycogen storage in 3T3-L1 adipocytes. J. Biol. Chem. 278, 30835-30842
Zhou XY, Shibusawa N, Naik K, Porras D, Temple K, Ou H, Kaihara K, Roe MW, Brady M.J., Wondisford FE. (2004) Insulin regulation of hepatic gluconeogenesis through phosphorylation of CREB-binding protein. Nat Med. Jun:10(6):633-7

Ou H, Yan L, Osmanovic S, Greenberg CC and Brady MJ (2005). Spatial reorganization of glycogen synthase upon activation in 3T3-L1 adipocytes. Endocrinology 146, 494-502

Yu C, Markan K, Temple KA, Deplewski D, Brady MJ and Cohen RN (2005). The nuclear receptor corepressors NCoR and SMRT decrease PPARg transcriptional activity and repress 3T3-L1 adipogenesis. J. Biol. Chem. 280, 13600-13605

Ou H, Yan L, Mustafi D, Makinen MW and Brady MJ (2005). The vanadyl (VO2+) chelate Bis(acetylacetonato)oxovanadium(IV) potentiates tyrosine phosphorylation of the insulin receptor. J. Biol. Inorg. Chem. (in press)

Greenberg CC, Danos AM and Brady MJ (2005). Central role for Protein Targeting to Glycogen in the maintenance of glycogen stores in 3T3-L1 adipocytes. Mol. Cell Biol. (in press)