Insulin Control of Glycogen Metabolism in Knockout Mice Lacking the Muscle-Specific Protein Phosphatase PP1G/RGL

In recent years, the generality that the activity of the type 1 serine/threonine protein phosphatases (PP1) is dictated by the associated noncatalytic subunits has emerged. These ancillary proteins are thought to target the catalytic component (C1) to distinct subcellular locales in proximity to substrates, to confer specificity, and to regulate activity (10, 21, 33, 41). To date, more than 30 C1-binding polypeptides have been identified that direct the enzyme to a variety of subcellular structures, including glycogen (6, 24, 25, 49, 59, 60), myosin (2), ribosomes (31), nuclei (4, 13), and neuronal structures (5). A subset of C1-binding proteins includes inhibitory proteins such as inhibitors 1 and 2 (48, 67) and DARPP-32 (46). Four C1-glycogen-targeting subunits are presently known. RGL, also called GM, was the first glycogen-binding subunit of PP1 identified (59), and the corresponding holoenzyme, PP1G/RGL, consists of the 124-kDa RGL protein (60) in association with C1. RGL is exclusively expressed in skeletal and cardiac muscle (37, 60). The NH2-terminal 240 amino acids contain binding sites for glycogen and C1 (64), whereas a hydrophobic region between residues 1063 and 1097 in the COOH terminus anchors the protein to membrane (45, 60). Of the other three glycogen-targeting subunits, GL, a 33-kDa polypeptide, is specifically expressed in liver (24), whereas PTG/R5 and R6 are ubiquitously distributed (6, 25, 49). All three share homology with the NH2-terminal region of RGL. The activity of liver PP1G/GL is controlled allosterically by glycogen phosphorylase (Ph) and by glucose-6-phosphate (G-6P) (1, 58), and expression of the GL subunit is downregulated in diabetic rats (23). Protein targeting to glycogen (PTG) interacts with glycogen-metabolizing enzymes (26) and has been implicated in insulin control of glycogen synthase (GS) (49). Overexpression of PTG in Chinese hamster ovary cells expressing the human insulin receptor increased basal and insulin-stimulated GS activity (49). Neither insulin nor forskolin induced detectable PTG phosphorylation, arguing against such a mechanism for PTG regulation. Adenovirus-mediated overexpression of GL, PTG, or RGL in primary rat hepatocytes results in basal activation of GS (12), but only in cells overexpressing GL or PTG was GS activated by insulin (27). Other studies have postulated a mechanism whereby PTG would affect PP1 activity by relieving inhibition by DARPP-32 (16). However, it has been shown that neither I-1 nor DARPP-32 is required for insulin activation of GS (53). Thus, the mechanisms for control of PTG- and R6-containing phosphatases are largely unknown. In mammals, the major stores of glycogen are in skeletal muscle and liver. Although glycogen in these two tissues performs different functions, both pools contribute to glucose homeostasis. Approximately 80% of postprandial, insulin-stimulated glucose uptake is stored as glycogen in skeletal muscle (56). This insulin-stimulated glycogen synthesis involves activation of both glucose transport and GS (39). Glycogen metabolism is controlled in large part by the coordinated regulation of the two enzymes responsible for its synthesis and breakdown, GS and Ph. GS is inactivated by a complex, multisite phosphorylation mechanism involving several protein kinases, including GS kinase 3 (GSK-3) (38, 52). The allosteric activator G-6P can restore full activity, so that the GS activity ratio in the absence and presence of G-6P (−/+ G-6P activity ratio) is used as a kinetic index of phosphorylation state. Ph is activated by phosphorylation of a single site by phosphorylase kinase (PhK). Insulin promotes dephosphorylation and activation of GS, with no significant effect on Ph. Despite extensive investigation over the last four decades, the exact molecular mechanism(s) by which insulin activates GS in skeletal muscle is not fully understood. Epinephrine causes phosphorylation of both GS and Ph, leading to their respective inactivation and activation so that glycogen is degraded. Dephosphorylation of the key regulatory enzymes, GS, Ph, and PhK, is believed to be catalyzed primarily by glycogen-associated phosphatases (59). Also, association of RGL with C1 has been shown to enhance activity towards these three glycogen-metabolizing enzymes (33). PP1G/RGL has been postulated to play a central role in both insulin and epinephrine control of glycogen metabolism in skeletal muscle, via phosphorylation of the RGL-targeting subunit (20, 47). Insulin control of PP1G/RGL would be mediated by the insulin-stimulated protein kinase ISPK or p90rsk which is itself regulated via the mitogen-activated protein kinase pathway (20). ISPK phosphorylates RGL at site 1 in vitro, enhancing the rate of dephosphorylation of GS and PhK, with no effect on the activity towards Ph. Treatment of rabbits with insulin was reported to increase the phosphorylation of site 1 (20). The hypothesis for control of PP1G/RGL by insulin was attractive, since this enzyme dephoshorylates GS at both the COOH- and NH2-terminal sites that are responsive in vivo to the hormone (38). However, several reports have shown that the mitogen-activated protein kinase pathway is not involved in insulin activation of GS (9, 40). Nevertheless, the results of these studies do not preclude the possibility that PP1G/RGL mediates insulin regulation of glycogen metabolism by other mechanisms. Newer models for the control of GS activity by insulin have centered on a role for the PI-3K pathway (17, 54) and control of GSK-3, an important GS kinase whose activity is known to be inhibited by insulin. Stimulation of PI-3K by insulin would activate Akt/PKB which phosphorylates and inactivates GSK-3. Another pathway activated by insulin involves mTOR, the mammalian target for the immunosuppressant drug rapamycin (9); rapamycin has been shown to oppose insulin-mediated activation of GS in muscle and 3T3-L1 adipocytes (9, 54). Since rapamycin does not block insulin-induced inactivation of GSK-3 (18), it is possible that mTOR could control GS phosphorylation via a phosphatase. To address the role of PP1G/RGL in the hormonal control of glycogen metabolism in a more definitive manner, we have generated RGL knockout mice. Analysis of this animal model clearly demonstrates that RGL has an important role in glycogen synthesis but is not essential for insulin activation of GS.